System for Separating a Cell Sample by Centrifugation and Column Chromatography While Maintaining Sterility

ABSTRACT

The invention relates to a system, comprising: a) a sample processing unit, comprising an input port and an output port coupled to a rotating container having at least one sample chamber, the sample processing unit configured provide a first processing step to a sample or to rotate the container so as to apply a centrifugal force to a sample deposited in the chamber and separate at least a first component and a second component of the deposited sample; and b) a sample separation unit coupled to the output port of the sample processing unit, the cell separation unit comprising separation column holder ( 42 ), a pump ( 64 ) and a plurality of valves (1-11) configured to at least partially control fluid flow through a fluid circuitry and a separation column ( 40 ) positioned in the holder, the separation column configured to separate labeled and unlabeled components of sample flowed through the column.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/745,688, which is the U.S. National Stage of PCT/IB08/03823, filedDec. 8, 2008, and having a §371(c)(1) date of Aug. 20, 2010, whichclaims priority to U.S. Provisional Application 61/012,361, filed Dec.7, 2007. The PCT application and U.S. National Stage application arehereby incorporated herein by reference in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

A variety of human diseases currently cannot be treated in asatisfactory manner with standard pharmaceuticals, proposing the use ofprimary human cells as an alternative or additional op-tion for thetreatment of various diseases. Those cellular therapy approaches usuallyrequire significant handling and processing of cellular products toseparate wanted from unwanted functions, for example depletion of cellsin an unwanted and potentially life threatening graft versus hostreaction or enrichment of cells involved in wanted graft versusleukemia/tumor effects.

Methods known in the art require a massive infrastructure in hospitalsto fulfill regulatory and safety requirements, including goodmanufacturing procedures compatible clean rooms and personnel tomaintain rooms, devices, production, quality control and qualityassurance procedures. Cellular products are usually processed utilizinga combination of different devices and disposables with manual transferof samples between those systems.

The current invention integrates various cell processing steps into asingle device and disposable, controlled in a fully automated process,eliminating the requirements for manual cell transfer, in-processcontrols, related risks to the cellular product, and risk reductionmeasures and thus provides a device and method for manufacturing ofcellular therapy products that are ready for direct use. The cellularproduct manufactured using the system of the present invention willtypically be ready for direct transfer into the patient.

The present invention is generally related to processing of biologicalmaterials. More specifically, the present invention provides systems,devices, and methods for the processing of biological materials toculture and/or separate components of a biological sample, and tofurther separate components of the sample by separation techniques,including application of magnetic separation.

Various techniques are known for separating components of a sample orbiological material that make use of separation techniques. Suchtechniques include but are not limited to pan-ning, magnetic separation,centrifugation, filtration, immunoaffinity separation, gravitationseparation, density gradient separation, and elutriation.

Immunoaffinity methods may include selective labeling of certaincomponents of a sample (e.g., antibody labeling) and separation oflabeled and unlabeled components. Magnetic separation methods typicallyinclude passing the sample through a separation column.

Magnetic separation is a procedure for selectively retaining magneticmaterials in a chamber or column disposed in a magnetic field. A targetsubstance, including biological materials, may be magnetically labeledby attachment to a magnetic particle by means of a specific bindingpartner, which is conjugated to the particle. A suspension of thelabeled target substance is then applied to the chamber. The targetsubstance is retained in the chamber in the presence of a magneticfield. The retained target substance can then be eluted by changing thestrength of, or by eliminating, the magnetic field.

A matrix of material of suitable magnetic susceptibility may be placedin the chamber, such that when a magnetic field is applied to thechamber a high magnetic field gradient is locally induced close to thesurface of the matrix. This permits the retention of weakly magnetizedparticles and the approach is referred to as high gradient magneticseparation (HGMS).

The use of HGMS in biological separations requires that the conditionsprovide a high yield with substantial purity. Accordingly, it would bedesirable to provide high gradient magnetic separators, devices andmethods that are relatively easy to construct and use, yet providemaximized and uniform magnetic field gradients and flow characteristicsduring use. It would be most advantageous if such magnetic separators,devices and methods could be used to perform a variety of cell sortingor assay procedures with the selection of the appropriate specificbinding member by which the target substance will be magneticallylabeled.

In many instances, separation methodologies must be performed underconditions that ensure non-contamination of the sample or maintainsterility. For example, many current clinical cell separation systemsneed to be operated in clean rooms of high quality in order to maintainsterility of samples. Often ensuring non-contamination is cumbersome,expensive and requires separate facilities and personnel, as well ascomplex procedures requiring extensive efforts to maintainreproducibility and sterility. Additionally, numerous processing andhandling steps (e.g., washing, volume reduction, etc.) must be performedseparate from the separation systems with subsequent introduction of theprocessed samples as well as attachment of fluids and reagents to thesystems, further complicating sterility compliance. As such, improvedmethods and systems are needed to ensure non-contamination of samplesand/or reducing the complexity and expense of sample processing.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a sample processing and separation systemincluding a sample processing unit and a sample separation unit.

In one embodiment, the invention provides improvements in high gradientmagnetic separation devices, apparatus and methods for the separation ofbiological materials. The subject devices, apparatus and methods allowsa greater specificity and efficiency in the isolation of particularcells, proteins, polysaccharides, and other biological materials, orother materials that are magnetic or are capable of a specific bindinginteraction with a magnetically labeled binding member.

A magnetic separation column is provided, having a nonmagnetic housingthat defines a separation chamber, and a fluid permeable matrix of e.g.metallic spheres within the chamber. The spheres form a closely stackedlattice, which creates substantially uniform channels for homogeneousflow during separations. A magnetic separator device may use theseparation column in conjunction with a prefilter device. The device maybe used in an instrument having a permanent or electromagnet for useduring separations, with an optional retractable arm for moving themagnet, pumping means for washing and separating the target material,and a microprocessor for controlling the separation fluid flow.

In one embodiment, the system comprises a) a sample processing unitcomprising an input port and an output port coupled to a rotatingcontainer (or centrifugation chamber) having at least one samplechamber, wherein the sample processing unit is configured to provide afirst processing step to a sample or to rotate the container so as toapply a centrifugal force to a sample deposited in the chamber andseparate at least a first component and a second component of thedeposited sample; and b) a sample separation unit coupled to the outputport of the sample processing unit, the sample separation unitcomprising a separation column holder, a pump, and a plurality of valvesconfigured to at least partially control fluid flow through a fluidcircuitry and a separation column positioned in the holder, wherein theseparation column is configured to separate labeled and unlabeledcomponents of sample flowed through the column.

It is preferred that the rotating container comprises a means fordetecting the progress of separation of the at least first component andthe second component of the deposited sample in the rotating container.

The means for detecting the progress of separation can be located suchthat light from a light source can at least partially penetrate throughat least a part of the sample that is being separated, and light passingthrough at least a part of the sample can be detected by a lightdetector.

Preferably, the means for detecting the progress of separation can belocated at the rotating container, essentially perpendicular to arotation axis of the rotating container.

It is further preferred that the means for detecting the progress ofseparation is positioned at the rotating container reaching essentiallyfrom an area adjacent to the rotation axis of the rotating container toan area adjacent to the perimeter of the rotating container.

The means for detecting the progress of separation in the rotatingcontainer can be a window, a prism or a mirror. The window, prism ormirror can be positioned to cover a channel formed in the lid or thebottom of the rotating container, wherein the channel is configured suchthat at least a part of the sample can flow into the channel duringcentrifugation. A window can also be used without a channel, i.e. theseparation of the sample is then detected through a window in the lid orthe bottom of the rotating chamber.

Preferably, the rotating container is configured such that it is usablefor culturing cells. In that case, the rotating container preferablycomprises at least one layer for growing cells thereon. The at least onelayer can be positioned perpendicular to a rotating axis. It ispreferred to ar-range a plurality of layers for growing cells thereon inthe rotating container.

The rotating container can be manufactured in a disposable form. It isalso preferred that the rotating container can be sterilized to allowcell processing without contamination.

In particular, the rotating container comprises or can be made of amaterial chosen from the group consisting of: plastics, polystyrol (PS),polysterene, polyvinylchloride, polycarbonate, glass, polyacrylate,polyacrylamide, polymethylmethacrylate (PMMA), polyethylenterephtala(PET), polytetrafluorethylen (PTFE), thermoplastic polyurethane (TPU),silicone The chamber can also be made of polyethylene (PE), collagen,chitin, alginate, hyaluronic acid derivatives, polyactide (PLA),olyglycolida (PGA) and their copolymers, polystyrol (PS), polysterene,polycarbonate, polyacrylate, ceramics, glass materials, likehydroxylapatite (HA), and calcium phosphate, and compositions comprisingone or more of the above mentioned materials.

Input and output ports typically comprise at least one sterile filter.

Furthermore, the invention relates to a method, comprising or containingthe following steps:

a) providing a system as described above and herein; b) performing afirst processing step to separate at least a first component and asecond component of a sample; and c) performing a sample separation stepto a processed component of the sample, the separation comprisingseparating labeled and unlabeled components from the processed samplecomponent. The processing step can also be performed after theseparation step.

Due to the presence of the rotating chamber in the system as describedabove and herein, it is preferred that the method comprises detectingthe progress of separation, in particular by detecting the formation oflayers of the sample, the change of pH value, and/or the change oftemperature.

The method can preferably be performed when the sample is a biologicalsample, in particular, blood or bone marrow.

Furthermore, the invention relates to a computer program, in particularwhen executed on a computer, for controlling a system as described aboveand herein, in particular for performing a method as described above andherein. The computer program can be stored on a storage means, such as afloppy disk.

The invention further relates to an integrated cell processinginstrument comprising a housing, at least one magnet unit for disposablemagnetic separation chamber, at least one drive for a disposablecentrifuge/culture chamber, various pinch valves arranged such thatdifferent ar-rangements of a disposable tubing set can be mounted ontothe instrument. Further, the instrument may comprise a user interfacewith integrated monitor and/or computer for storing and performingdifferent cell processing operations. For this purpose, at least onepump driver can be operated using the computer. An optical detectionsystem for a centrifuge for measuring optical densities in differentpositions in the chamber can be present. The centrifuge chamber can bedisposable and located in a temperature controlled area. Means foradjusting the temperature of the fluid moving to the chamber can bepresent (heater, cooler). A cell culture camera may be positioned at therotating container, preferentially located at bottom, possibly withmeans for adjusting the focus of the camera.

The invention further relates to disposable sterile tubing set forsterile cell processing for use with the system of the invention.Typically, a sample bag or port for starting cells is provided. Thetubing and/or the system can be configured to allow direct transfer ofcell from patient to a sample bag. Further, the following can be part ofthe tubing and/or system: a different input port for buffer cell culturemedia, at last one input port magnetic labeling reagent, input portstypically with sterile filter(s), an output port for waste, and/or anoutput port for cells. The output port can be directly connected withcell freezing bags. Further, an output port can be directly connectedwith container for transfusion and/or injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an exemplary separation orprefiltration column of the present invention.

FIG. 2 depicts separation and prefiltration columns, together with thesample and collection containers, interconnected by a series of fluidpathways or fluid circuits. The figure also shows the positioning ofvalves and a peristaltic pump that is used in the preferred embodimentof the separation system.

FIG. 3 depicts a computer-controlled unit on which sterile columns,disposable tubing and storage and collection containers, as illustratedin FIG. 2, are appended. In a preferred embodiment, thecomputer-controlled unit contains a magnet, valves and peristaltic pump.

FIG. 4 depicts the flow channel formed between three connecting spheres.

FIGS. 5 through 7 illustrate a system according to an embodiment of thepresent invention.

FIG. 8 illustrates a chamber of a processing system, according to anembodiment of the present invention.

FIG. 9 illustrates a chamber of a processing system, according toanother embodiment of the present invention.

FIG. 9A illustrates a chamber of a processing system, according to anembodiment of the present invention.

FIG. 10 shows a cross-sectional view of a processing chamber, accordingto an embodiment of the present invention.

FIG. 10A illustrates a top plan view of a processing chamber, accordingto an embodiment of the present invention.

FIG. 11 shows a cross-sectional view of a processing chamber, accordingto another embodiment of the present invention.

FIG. 11A illustrates a focused view of a portion of a processing chamberas shown in FIG. 11.

FIG. 12 shows a cross-sectional view of a processing chamber, accordingto yet another embodiment of the present invention.

FIG. 12A shows a view over the bottom of the process chamber with anopening for gas delivery and a bonded hydrophobic membrane.

FIG. 12B shows the bottom of the chamber with spiral channels foraeration through membrane bonded to the bottom of the chamber.

FIG. 13 illustrates a system according to an embodiment of the presentinvention.

FIG. 14A through 14N illustrate an exemplary sample processing methodaccording to an embodiment of the present invention.

FIG. 15 illustrates an aeration device that can be part of the systemaccording to the invention.

FIG. 16A shows a gas-mixing chamber that can be part of the systemaccording to the invention.

FIG. 16B shows the bottom part of the gas-mixing chamber that can bepart of the system according to the invention.

FIG. 17 shows a view of the inside of a lid for a rotating chamber witha channel or gap in which the sample flows during centrifugation, with ameans for detecting the progress of separation of the sample in the formof a prism.

FIG. 18 shows the path of light through the sample by means of a prism.The prism (double prism) is configured such that light from a lightsource can at least partially penetrate through at least a part of thesample that is being separated through centrifugation, and light passingthrough at least a part of the sample can be detected by a lightdetector.

FIG. 19 shows a double prism that is part of a rib located at the lid ofthe rotating chamber.

FIGS. 20 to 29 show the results of experiments performed with a systemaccording to the present invention, using a method according to thepresent invention.

FIG. 20 shows unprocessed bone marrow (A, C) and CD133 selected cells(B, D).

FIG. 21 shows unprocessed apheresis product (A, B) and CD14 enrichedproduct (C, D).

FIG. 22 shows unprocessed apheresis harvest (left) and enriched PDCs(right).

FIG. 23 shows CD4 selection using the present invention, unprocessedbuffy coat (left), CD4 enriched target cells (right).

FIG. 24 shows Buffy coat cells before (left) and after (right) CD8depletion using the present invention.

FIG. 25 shows growth of K562 cell line in centrifuge chamber.

FIG. 26 shows unprocessed bone marrow (left) and CD34 or CD133 selectedcells after direct elution in 20 ml (right).

FIG. 27 shows unprocessed bone marrow (left) and CD34 or CD133 selectedcells after direct elution in small volume of 6 ml (right).

FIG. 28 shows unprocessed bone marrow (left) and CD34 or CD133 selectedcells after final volume reduction by filter (right).

FIG. 29 shows unprocessed bone marrow (left), CD34 or CD133 selectedcells after direct elution in 20 ml (middle) and after final volumereduction by AutoMACS column.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides sample processing systems that integrateboth sample separation systems and sample processing techniques. Asystem can include a sample processing unit configured to performcertain processing steps prior to separation methods, such as magneticbased separation, cell culture or cell manipulation. As such, thepresent invention can include a combined sample processing system andsample separation system. Sample processing systems or units can providesample processing such as cell culturing, washing, preparation,incubation, labeling and the like. Additionally, sample processingsystems/units can include centrifugation based separation techniques,where a centrifugal force is applied to a sample so as to separate atleast a first component and a second component from a sample.

Thus, a system of the present invention will typically include both asample processing unit and a sample separation unit. The system may alsocomprise or contain a plurality of sample processing units and/or aplurality of sample separation units. The combined processing/separationsystem of the invention can include a closed system that can beprogrammed to automatically perform a variety of complex cell processingsteps, including density based separations, immunoaffinity separation,magnetic including immuno-magnetic separations, cellcultivation/stimulation/activation, washing or final formulation steps.For this purpose, the system may be controlled by a computer programthat can be run on a computer. The cell processing steps also mayinclude delivery of certain substances to the cells, includingcytokines, genetic materials as DNA, RNA, viruses, transcriptionfactors, antigens or other chemical substances.

The invention provides a system that minimizes errors of the user,maintains sterility, per-forms complex cell processing steps with littleor no manual interaction required, and minimizes operator exposure whenprocessing infectious material. Processing at bedside or in a surgicalroom is possible. The device can be operated while connected to apatient from which a sample is being obtained or to whom a processedsample or fractions thereof are administered. E.g., bone marrow obtainedfrom a patient may be processed directly into an input bag of the tubingset. From there, the e.g. bone marrow can be processed, i.e. separatedinto at least two components. At least one of these components may beadministered to the patient, possibly after processing the component ina suited way.

Density based separation processes performed with the sample processingunit of the inventive system including, without limitation, cellwashing, buffy coat generation, density based separation using densitymedia, e.g. Ficoll™, Percoll™ (GE Healthcare), separation based ondensity labeling (e.g. Rosettesep (Stem Cell technologies), or otherdensity labeling procedures), separation due to speed of sedimentatione.g. removal of thrombocytes, elutriation, cell adhesion, and the like.Additional processing steps/techniques that can be performed with asample processing unit of the inventive system include cell cultureincluding expansion, stimulation, differentiation, re-differentiation,antigen loading, transfection, transduction, culture, adherent or insuspension, multi-cell type multilayer or mixed, culture in standstillor with sheer forces or mixing. Input materials include but are notlimited to blood, leukophere-sis, bone marrow, liposuction, milk, anybody fluid, cells from tissue e.g. cells from various organs, tumorcells, single cells, cell clumps, cell aggregates, tissue dissectedmechanically or in conjunction with enzymes.

As stated above, a system of the invention will typically include asample processing unit/system and a sample separation unit/system.Sample separation systems will typically include magnetic basedseparation systems. One such magnetic based sample separation systemthat can be included in the system of the present invention includesmagnetic separation sys-tems/processes described, for example, on theworld wide web at the hyperlink “MiltenyiBio-tee.com”, and can be usedfor almost any cell type. Exemplary magnetic separation systems, whichis described in part below, are also described for example in theEuropean Patent Specification EP 0 869 838 B1 and in U.S. Pat. No.5,691,208, which are hereby incorporated by reference.

Improved magnetic separators, devices and methods for magneticseparation procedures are provided and described in EP 0 869 838 B1,which can be included in a system of the present invention. The matricesof the magnetic separators provide uniform pores or channels that reducethe entrapment of air or non-target substances, decrease the loss oftarget substances due to mechanical disruption.

Biological substances, such as target cells from various systems andorgans, are magnetically labeled with a suitable specific bindingmember, and isolated using the devices and methods of the presentinvention. The isolation of multipotential cells such as hematopoieticstem or progenitor cells is of particular interest. While hematopoieticcell separation is used herein to provide examples of cell separationprocedures, the present invention may be applied to a wide range of celltypes or other biological substances.

Cells processed using the present invention can be used for variouspurposes, e.g. in treatment of diseases, utilizing their potential toproliferate and to differentiate as well as their biological function(s)in living entities, e.g. blood or tissue.

Applications of cells that may be processed using the present inventioninclude, but are not limited to

-   -   graft engineering, e.g. in conjunction with for stem cell        transplantation    -   organ transplantation    -   cancer treatment including but not limited to leukemia including        acute myeloid leukemia, chronic myeloid leukemia and solid        tumors such as renal cell carcinoma, breast cancer, melanoma,        pancreatic cancer    -   treatment of refractory autoimmune diseases such as systemic        lupus erythematosus or systemic scleroderma, type 1 Diabetes,        multiple Sclerosis    -   cellular therapy including but not limited to directly utilizing        effector cells    -   treatment of infectious diseases    -   tissue regeneration including, but not limited to myocardial        infarction, liver damage or neurodegenerative diseases, and    -   tolerance induction including but not limited to transplantation        or autoimmune disease.

Processing methods using the present invention can be combined ofvarious basic operations including cell washing, media exchange, cellconcentration, incubation of cells with various substances (includingantibodies, cytokines, magnetic separation reagents, media), magneticcell separation, filtration, and cell culture.

Magnetic cell separation methods can comprise both enrichment anddepletion procedures (Bosio et al. in “Engineering of Stem Cells”,Springer Ser. No. 03/2009). If target cells can be identified based onsurface proteins, target cells can be enriched to high purity. In somesituations, non-target cells can be identified based on their unwantedfunctional characteristics within a specific clinical context. Thesenon-target cells can be removed from the cellular product, resulting ina heterogenous mixture of different target cells.

Cell products processed by the present invention for graft engineeringapproaches can be enriched for CD34, CD133 or depleted for CD3, CD3 andCD19, CD6, CD4 and CD8, T Cell Receptor alpha/beta (TCR alpha/beta) orCD3/CD19/CD16/CD14, resulting either in enriched stein cell preparationsor stem cells supplemented with other immune cells such as NaturalKiller cells and dendritic cells.

Cell products processed by the present invention for cellular therapyapproaches can be enriched e.g. for CD14 (monocytes), CD56 (naturalkiller cells), CD335 (NKp46, natural killer cells), CD4 (T helpercells), CD8 (cytotoxic T cells), CD1c (BDCA-1, blood dendritic cellsubset), CD303 (BDCA-2), CD304 (BDCA-4, blood dendritic cell subset),NKp80 (natural killer cells, gamma/delta T cells, effector/memory Tcells), “6B11” (Va24/Vb11; invariant natural killer T cells), CD137(activated T cells), CD25 (regulatory T cells) or depleted for CD138(plasma cells), CD4, CD8, CD19, CD25, CD45RA, CD45RO. Natural killercells, natural killer T cells, T cells and their subsets can be utilizedas effector cells in donor lymphocyte infusion approaches to eliminatevirus infected cells, tumor cells or bacteria. Dendritic cells, eithergenerated from monocytes in cell culture or directly isolated, can beused to “vaccinate” patients to promote antigen specific and naturalimmunity against virus infected cells, tumor cells, bacteria, and/orfungi.

Advantageously, the present invention allows for manufacturing ofcellular products by sorting for two or more parameters that can beperformed in a single tubing set without requirements to transfer thecell suspension from one single use tubing set to another, thus avoidingpotential harm to the cell product (infection, contamination, increasedtemperature). Two pa-rameter sorting applications include generation ofhighly enriched regulatory T Cells (cell product is first depleted forCD8 and/or CD19 and/or CD49d and subsequently enriched for CD25), ofhighly enriched natural killer cells (CD3 depleted, CD56 enriched) andof highly enriched blood dendritic cell subsets (CD19 depleted, CD1cenriched).

Tissue regeneration approaches usually utilize progenitor cells fromblood, bone marrow or tissue to (re-)vasculize tissue, promotegeneration of novel tissue or directly provide in vitro generatedtissue. Cells utilized can include cell products enriched for CD133,CD34, CD271 (LNGFR; mesenchymal stem cells), anti-MSCA-1 (W8B2;mesenchymal stem cells), CD144 (endothelial cells).

It is a specific and novel characterization of the current inventionthat manufacturing of cellular products by cell separation and culturecan be performed in a single tubing set without requirements to transferthe cell suspension from one single use tubing set to another. Inparticular, the current invention can be used to obtain Stem cells,T-Cells, dendritic cells, NK-cells, B-cells, monocytes, cells positivefor a particular marker, such as CD133, CD34, CD3, CD4, 8, 56, 19, 14,CD141 (BDCA-3), CD303 (BDCA-2), CD304 (BDCA-4), CD144, CD1c (BDCA-1),NKp46, NKp80, CD45RO, CD45RA, CD137, CD25, or CD138.

Composition/Formulation:

It is a specific and novel characterization of the current inventionthat cell products manufactured by basic operations as described abovecan be composed for direct clinical use. Methods known in the artrequire manual post-processing of engineered cell products to adapt itto clinical requirements. With the system of the present invention, thecell product can directly be formulated for immediate use. Formulationsteps include: adjustment to a desired volume or cell concentration,exchange of processing liquids by injectable liquids, addition ofstabi-lizers (such as autologous plasma or serum, serum albumins, otherproteins or synthetic poly-mers) or adjuvants, supplementation withcryoprotective agents such as DMSO for subsequent storage, drawing ofretain samples for quality control, delivery to combinations of bags orsyringes for infusion. Some components of the final formulation e.g.plasma, platelets, or platelet components may be derived from theoriginating sample.

The magnetic separation system of the present invention can be used tomagnetically label and isolate any desired target substance. Ofparticular interest is the separation of a specific component from acomplex mixture. The separation system of the present invention hasgreat versatility, in that almost any target substance may be separatedonce a specific binding member is available. The target substance oranalyte may be any member of a specific binding pair, or a substanceassociated with a member of a specific binding pair. As an example, acell surface antigen-antibody binding pair may be used to isolate theantigen itself, cells that express the antigen, a particular organelleinvolved in processing of the antigen, etc. The devices and methods ofthe present invention are also advantageously applied to diagnostictechniques involving the binding of a receptor and ligand, such asimmunoassays, and the like.

In its simplest form, a cell separation system of the present inventionhas two main components: a magnetic separator and a cell separationreagent. A schematic diagram of a magnetic separator device is given inFIG. 1. The diagram shows the general construction of the separator andthe uniform fluid passage that results from the use of a matrix ofmetallic spheres. FIG. 2 depicts a more complex separation device,including the general positions of fluid passages, collection andstorage containers and the separation column. The fluid circuitry can beconstructed with integrated valves, or the valves may be appliedexternally to the fluid pathways.

An optional third component to the preferred cell separation system is acell separation instrument. FIG. 3 depicts a cell separation instrument,preferably computer-controlled, which may incorporate the valvestogether with a magnet, pump and keyboard control. A device similar tothat of FIG. 2, constructed without valves, may be mounted directly ontothe instrument of FIG. 3 for use in the automated separation of targetcells.

The cell separation reagent, which may also be referred to as aconjugate, antibody/magnetic particle reagent or magnetic label,includes a magnetically responsive material bound to a specific bindingmember. There are many well-known magnetically responsive materials usedin magnetic separation methods. The present invention involves the useof magnetically responsive particles or microparticles. Suitablemagnetic particles are described in Molday U.S. Pat. No. 4,452,773, andin the European Patent Specification EP 452342 B, which are herebyincorporated by reference. Colloidal sized particles, such as thosedescribed in Owen U.S. Pat. No. 4,795,698, and Liberti et al. U.S. Pat.No. 5,200,084, are also suitable.

The term “specific binding member” as used herein refers to a member ofa specific binding pair, i.e. two molecules, usually two differentmolecules, where one of the molecules through chemical or physical meansspecifically binds to the other molecule. The complementary members of aspecific binding pair are sometimes referred to as a ligand andreceptor. In addition to antigen and antibody specific binding pairs,peptide-MHC antigen and T cell receptor pairs; alternative specificbinding pairs of interest include biotin and avidin or streptavidin;carbohydrates and lectins; complementary nucleotide sequences (includingnucleic acid sequences used as probes and capture agents in DNAhybridization assays); peptide ligands and receptor; effector andreceptor molecules; hormones and hormone binding protein; enzymecofactors and enzymes; enzyme inhibitors and enzymes; secretion markers,as described in International application PCT/US93/10126 (herebyincorporated by reference); autologous monoclonal antibodies, and thelike. The specific binding pairs may include analogs, derivatives andfragments of the original specific binding member. For example, anantibody directed to a protein antigen may also recognize peptidefragments, chemically synthesized pep-tidomimetics, labeled protein,derivatized protein, etc. so long as an epitope is present.

Immunological specific binding pairs include antigens and antigenspecific antibodies or T cell antigen receptors. Suitable antigens maybe haptens, proteins, peptides, carbohydrates, etc. Recombinant DNAmethods or peptide synthesis may be used to produce chimeric,trun-cated, or single chain analogs of either member of the bindingpair, where chimeric proteins may provide mixture(s) or fragment(s)thereof, or a mixture of an antibody and other specific binding members.Antibodies and T cell receptors may be monoclonal or polyclonal, and maybe produced by transgenic animals, immunized animals, immortalized humanor animal B-cells, cells transfected with DNA vectors encoding theantibody or T cell receptor, etc. The details of the preparation ofantibodies and their suitability for use as specific binding members arewell known to those skilled in the art.

For brevity, the separation system will mainly be described in terms ofits ability to specifically select and separate a defined population ofcells (target cells) from a mixed cell population, such as peripheralblood, bone marrow, blood from the umbilical cord or placenta, fetalblood or a leukapheresis product. It will also be appreciated that sometissues may be dis-rupted into a single cell or monodisperse suspensionto allow isolation of a particular cell subset, such as the separationof tumor infiltrating lymphocytes from a tumor mass, the separation ofislet cells from kidney tissue, etc. For example, different cell typesmay be labeled with a specific antibody to allow cell purging and/orcell enrichment. The target cell population is generally identified by aspecific binding member, as described above, which selectively binds toa cell surface antigen present on the target cells. It should beunderstood, however, that the subject apparatus and method is notlimited to such uses.

For simplicity, the specific binding member will be exemplified hereinby an antibody. The antibody may be directly or indirectly bound to amagnetic particle. If the antibody is directly bound to the magneticparticle, then the target cell population is magnetically labeled whenthe antibody binds to the cell surface antigen. If the antibody isindirectly bound to the magnetic particle, then the target cellpopulation is susceptible to magnetic labeling when the antibody isbound to the target cells. The antibody-bound cell population isactually labeled by further contacting the cells with a specific bindingmember for the antibody, where that specific binding member is itselfbound to a magnetic particle. The target cells, identified by suchmagnetic labeling, are then separated from other cells by means of amagnetic field. For example, a specific binding member such as avidincan be conjugated to a magnetic particle where the avidin binds to abiotinylated antibody that in turn specifically binds to the targetcells.

The specific binding member may be directly attached to the magneticparticle. This may be accomplished by means of reactive groups on thespecific binding member and magnetic particle themselves. Alternatively,the specific binding member and magnetic particle may be joined by meansof a coupling agent or linker. The terms “coupling agent” or “linker”,as used herein, include various bifunctional crosslinking or couplingagents, i.e. molecules containing two reactive groups or “ends”, whichmay be separated by a spacer.

Conventional high gradient magnetic separation matrices are typicallyprepared from materials such as wires, metal-coated fiber or steel wool.In the improved magnetic separation device of the present invention, thegradient-intensifying matrix of the high gradient magnetic separator isformed from small spheres of magnetically susceptible or ferromagneticmaterial. Such materials include, but are not limited to iron, steel,cobalt nickel, and other ferromagnetic rare earth metals or alloysthereof. For example, the matrix material may include ferromagneticmetal spheres such as iron spheres (e.g. MARABU Balls, KugelfabrikSchulte & Co., Wermelskirchen, Germany). Many different methods ofmanufacturing spheres are known. Usually the spheres have an averagediameter ranging from about 0.2 to 1.5 mm for the separation of largecells or cell complexes, and about 0.05 to 0.2 mm diameter forsubcel-lular material. Preferably, the spheres have an average diameterranging from about 0.2 to 0.5 mm, and most preferably, the spheres areselected to have an average diameter ranging from about 0.2 to 0.3 mm.It is desirable that the size of spheres be relatively homogeneous,usually varying not more than about 15% from the average size, moreusually by not more than about 10%, and preferably by not more thanabout 5%.

The substantially symmetrical spherical shape and substantially uniformsize of the spheres are desirable for the construction of a magneticseparator matrix, as the spheres can assume a lattice configurationwherein the gaps between the spheres form regular channels or pores inthe matrix. The lattice configuration is a patterned framework ofspheres that forms channels of regular size between adjacent spheres andthroughout the matrix. Upon the application of a magnetic field to theseparator, magnetic field gradients are created in the gaps between thespheres. The uniform size, and therefore spacing, of the spheresprovides for a substantially uniform magnetic gradient throughout thematrix, and substantially uniform fluid flow characteristics. A flowchannel is depicted in FIG. 4. The dimensions of the channel can bedescribed by the maximum size ball or particle that would fit betweenthe matrix spheres. With reference to FIG. 4, the geometricalrelationship is r=about 0.155 R. It will be appreciated from theteachings of the present invention that the channel size may be adjustedto an average diameter optimal for the desired separation process byvarying the size of the spheres that are used to form the matrix.

The spherical shape provides for the formation of a substantially stablematrix structure when the spheres are packed within a housing thatdefines a separation chamber. As described in detail below, the matrixis also coated with a substantially fluid impermeable material such as aplastic polymer. On application of a plastic polymer coating, the tightgaps between the spheres are closed, resulting in a hydrodynamicallyoptimized matrix. The resulting ferromagnetic matrix will usually occupyabout 60% to 75% of the total volume of the separation chamber, and willbe permeable to fluids. The impermeable coating will occupy about 1 to5% of the total volume. The free volume will range from about 20% to 40%of the total separation chamber volume. In a preferred embodiment, thetotal matrix will occupy about 75% to 80% of the total volume of theseparation chamber.

FIG. 1 presents a schematic of an exemplary separation column. Thespheres are not depicted at scale, but so as to better depict theformation of a three dimensional fluid permeable matrix 6. The spheresare packed in a housing 4 which is made of a non-magnetic material. Themagnetic separator housing serves as the body of the separation column,and the interior of the housing defines a separation chamber. Housingsof various lengths, shapes and diame-ters are advantageously made ofplastic. Suitable nonmagnetic materials for the construction of amagnetic separator housing include stainless steel, glass, plastic, etc.

In one preferred embodiment, the magnetic separator housing is a plasticthat the matrix coating will adhere to, allowing improved hydrodynamicproperties at the boundary of the matrix and housing. It will beappreciated that the coating material and housing material will beselected for compatibility with one another, e.g. a lacquer coating mustbe selected that will not result in non-adherent plastic debrisaccumulating in the column. Various mechanisms are known by whichmaterials adhere to each other, and may be exploited for this purpose.Con-veniently, the selection for compatibility may be made based on thesolvent used in conjunction with the lacquer. The solvent will beslightly reactive with the plastic housing, such that the lacquer in thesolvent will adhere, but not so reactive that the structural integrityof the column is compromised during the lacquer curing process. Forexample, the coating material may be selected to include a solvent thatcauses a slight dissolution of the interior of the plastic housing. Uponcuring, the plastic rehardens thereby bonding or sealing the coatingmaterial and housing material to each other. One of skill in the artwill appreciate that information regarding such reactivity is generallyavailable. Exemplary of a suitable solvent and housing combination ismethylethylketone and the plastic ULTEM® (General Electric).

Preferably, each of the materials selected for the construction ofseparation column 2 will also be compatible with sterilizationprocedures. Preferably, the housing is cylindrical in shape tofacilitate the flow of sample through the separation chamber as well asthe formation of three-dimensional matrix 6 within the housing. Thewalls of the housing preferably have a thickness of about 1 to 3 mm. Theseparation column has inlet 12 and outlet 14 ports for the introductionand discharge of fluids. Generally, the inlet and outlet ports arenarrow structures relative to the main body of the housing. Thisfacilitates the attachment of the separator to further fluid circuitryin a separation system and advantageously maintains the device as aclosed system. The inlet and outlet ports may be positioned at differentsites than as depicted in FIG. 1, but it will be appreciated that theoverall structure of the separator will preferably provide a separationchamber having the fewest bends or corners which might otherwise slowfluid flow or create spaces where sample might accumulate.

At the inlet and outlet of the column, the column is constructed to havea feeding mechanism ensuring optimal homogeneous distribution and flowthrough the matrix. The distribution mechanism is comprised of thevolume in front of cap layer 8 and the cap layer itself, which serves asa flow resistor. The distribution volume (in milliliters) may be definedrelative to the width of the column (in millimeters), usually having aratio of about 0.1 to 10. The chamber volume in front of base layer 10,as well as the base layer itself, also serves as feeding mechanism forfluids passing into the chamber via the outlet port.

It is preferable to have column dimensions where the diameter to lengthratio is at least 0.2 to 1. The actual dimensions of the column willdepend on the material being separated, and the desired flow rate forseparation. The column dimensions will provide a chamber that willaccept a matrix having an adequate surface area to create sufficientmagnetic field gradients in the separation chamber and permittingefficient retention of magnetically labeled material. The volumenecessary for a given separation may be empirically determined, and willvary with the size, antigen density on the cell surface, antibodyaffinity, etc. As an example, a sectional area of 3 cm² allows a flowrate of 5 to 40 ml/minute. The binding capacity of a 2×4 cm matrix isapproximately 10⁹ labeled cells.

To facilitate the manufacture of the separation column, base layer 10 ofnonmagnetic porous material is positioned in the housing such that whenthe ferromagnetic spheres are placed in the chamber they do not passthrough outlet port 14. Suitable porous materials for the formation ofthe base layer include, but are not limited to, porous plastic, sinteredmetals or glass, grids, etc. For example, various porous frits availablefrom Porex Singwitz, Germany may be used. Usually, the porous materialwill have a pore size of about 20 to 200 μm, preferably of 50 to 150 μm.A suitable pore size will be selected according to the dimensions of thetarget substance and the make up of the sample material. In addition,the pore size will not be so large as to allow the spheres to fill theporous openings of the layer material. Following the insertion of thespheres into the chamber, the housing may be shaken or vibrated tofacilitate the settling of the spheres into a more uniformconfiguration. Optionally, cap layer 8 of non-magnetic porous materialis positioned in the housing over the matrix to maintain the uniformconfiguration of the matrix during storage, handling and use. Pressuremay also be applied to cap layer 8 to more firmly pack the sphereswithin the chamber. Upper portion 16 of the separation column, whichincludes inlet port 12 in this embodiment, is then positioned on the topof housing 4 and is attached to the housing. For example, when usingplastic materials, upper portion 16 might be glued or ultrasonicallywelded to housing 4 to complete the formation of the separation column.Following the completion of the housing, the matrix is coated.

With reference to FIG. 1, a coating is applied to the fluid permeablematrix described above. The coating is selected to be substantiallyimpermeable to ions, and therefore protects the metallic matrix materialfrom corrosion as well as inhibiting the escape of cations from thematrix, which might damage the cells. In addition to the formation of animpermeable protective layer over the matrix material, a completecoating of the matrix closes the gaps between spheres, providing bothmechanical stability to the matrix, and a hydrodynamically optimizedmatrix. Such mechanical stability is particularly advantageous when thematrix is formed from small spheres of magnetically susceptible orresponsive metal, as described above. A coating material such as alacquer coating may be flowed into inlet port 12 of the separationcolumn. The lacquer flows through cap layer 8, matrix 6 and base layer10, thereby coating the porous surface of each component. Excess lacqueris allowed to pass from the chamber outlet port 14. The coatedseparation column may be centrifuged to further expel excess coatingmaterial from the chamber. The coating is then allowed to dry. Theseparation column may be heated to further promote the drying of thecoating. For example, the coated separation column may be placed in anoven at 110° C. for four to five hours followed by continued drying atroom temperature for three to seven days.

Upon drying, the coating hardens, thereby providing mechanical supportto the matrix. Not only does this mechanical support aid in maintainingintegrity of the matrix during the storage and handling of theseparation column, it also provides the matrix with a rigid structurewhich does not exhibit significant elasticity. This rigidity isadvantageous because the matrix might otherwise be deformed upon theapplication of an external magnetic field to the separation column. Theapplied magnetic field strength of the external magnetic means istypically within a range of about 0.1 about 1.5 Tesla, and morepreferably between about 0.2 to about 0.8 Tesla. The field should begreat enough and the distance between the magnet and the separationcolumn should be small enough to induce intensified magnetic fieldgradients within the matrix. To maintain uniform magnetic gradients inthe separator, the matrix material should move or shift in the chamberupon the application of the magnetic field. The spherical metalliccomponents, the housing and the coating are advantageously combined inthe present invention to provide an improved matrix with sufficientrigidity to resist substan-tial deformation when the separation columnis placed within a magnetic field.

It is preferable to coat the matrix while the spherical metalliccomponents are within the separation column housing. Coating the matrixwithin the housing avoids disrupting the matrix after the coating hasbeen applied. Moreover, the matrix within the housing serves to fill orseal small void spaces, interstices crevices formed near the contactpoints between the spheres, as well as between the spheres and housing,while simultaneously providing a uniform surface to the channels orformed by the separated points of the spheres. These channels or poresresult in the permeability of the matrix. By sealing the void spaces,there is a decrease in the areas where cells or other solid componentsof the sample might wedge or become physically en-trapped, even in theabsence of a magnetic field.

In the completed separation column, the selection of matrix coatingmaterials will preferably result in channels or pathways through thepermeable matrix having an average diameter ranging from 20 to 60 μm andan occupying of about 60% to 80% of the total volume of the separationchamber. For example, a separation column for the separation of bloodcells may have a final coated-channel size averaging 20 μm, with thematrix occupying approximately 80% of the total volume of the chamber.

Following the preparation of the substantially impermeable coating, thematrix and other interior surfaces of the separation chamber arepreferably further treated by the addition of a hydrophilic materialsuch as polyvidone (BASF, Ludwigshafen, Germany). Other suitablehydrophilic coating materials include, but are not limited to,polyvinylpyrrolidine, polyethylene glycol, hydroxyethyl starch, andhydrophilic coatings, such as acrylamides, surfactants orde-tergent-type wetting agents, and biological material including, butnot limited to, heparin and human serum albumin. The interior surface ofthe separation chamber may also be made hydrophilic by plasma or coronaetching of the surface. The hydrophilic coating provides the interiorthe separation column and the fluid permeable matrix with a readilywettable surface. By enhancing the wettability of these surfaces, theintroduction of fluid into the separation column will produce a uniformfluid front as it passes through the chamber. This in facilitates theremoval of air bubbles from the permeable matrix and other void space inthe separation chamber. It is desirable to maintain the separationcolumn and other device components as a closed system substantially freeof air during the separation process. The presence of air in the systemduring the separation of target cells affects the interior surfacetensions and unventi-lated areas, which can lead to cell destruction.

Referring to FIG. 2, which depicts a separation device, separationcolumn 40 may be pre-ceded by a prefiltration device. The figure depictsthe prefiltration device as a column 30, however it will be understoodthat other configurations, such as prefilter, may find use. Theprefiltration column is generally a three dimensional structure that maybe substantially identical to the separation column in terms of itsstructural composition. The prefiltration column, however, may havedifferent dimensions, the matrix may be made of spheres having adifferent composition, e.g. a non-ferromagnetic material, or the matrixmay be made of spheres having a different diameter from those used inthe separation column, thereby providing a pore or channel sizedifferent from that found in the separation column. In one embodiment,the prefiltration column is identical to the separation column. Passageof the sample through the prefiltration column serves to trap and removefluid components that are not desired in the final separation product.For example, in blood cell separations, “sticky” cells such asmonocytes, granulocytes and platelets may be removed from the cellsuspension by the prefiltration column. Alternatively, the prefiltrationcolumn may be constructed to have an average pore size that is smallerthan that found in the separation column. For example, the pore size ofthe permeable matrix may be selected to remove large tumor cells fromthe sample prior to the fluid's passage through the separation column.The passage of the fluid sample through the prefiltration column mayalso serve to break apart aggregates, such as cell aggregates, that mayexist in the fluid. Moreover, because the prefiltration column containsmaterials substantially identical to those of the separation column,those sample components that might non-specifically bind to theseparation column are advantageously caught by the prefiltration column.Thus, the prefiltration column reduces the possibility of fouling theseparation column during the separation process, and it reduces thecollection of unwanted cells or fluid components in the final separationproduct.

Referring to FIG. 2, a preferred embodiment of separation device 25 isdepicted. Sample container 81 is connected to an optional suspensionfilter 35. The suspension filter may be used to remove unwantedparticulate components from the fluid sample and is selected to have apore size sufficient to remove particulates above a certain size. Forexample, the suspension filter may be a Pall Filter (Pall SQ40S; PallBiomedical, Inc., Puerto Rico) having a pore size selected to removeparticulates larger than 40 μm, such as cell clots and clumps inhematopoietic cell samples. The suspension filter is connected by fluidpathway two 12 to inlet port 32 of prefiltration column 30.

Outlet port 34 of the prefiltration column 30 is connected by fluidpathway five 15 to inlet port 42 of separation column 40 to which themagnetic field will be applied in the course of the separation process.Outlet port 44 of the separation column is connected by fluid pathwayeight 18 to distribution channel 88 which leads to product collectioncontainer 83, final wash waste container 84 and unlabeled samplecontainer 85. Separate fluid pathways nine 19, ten 20, and eleven 21lead to these containers, respectively.

This separation device further includes a wash or buffer container 80and an initial wash waste container 82, which are connected by fluidpathways one 16 and three 17, respectively, to fluid pathway two 12.Buffer container 80 is also connected via wash or buffer line 90 (fluidpathway six) to distribution channel 88. The buffer line is furtherconnected to fluid pathway five 15 by means of fluid pathway four 14.The fluid pathways, containers, filters and columns may be coupled toone another by means of any suitable means such as standard spikes, Luerlocks, male-female connectors, T-connectors, Y-connectors and 4-wayconnectors or other fittings as are commonly used in intravenoussolution delivery sets.

Fluid flow through the fluid circuitry of the separation device can becontrolled by means of valves placed within the fluid pathway(s). Fluidpathways one through eleven are associated with corresponding valves onethrough eleven (1-11). The valves may be inside the pathways themselvesor may be external to the pathways. The fluid flow may also becontrolled by a pump. For example, when the fluid pathways are made of aflexible material, such as flexible tubing, suitable valves for thecontrol of fluid transport include pinch valves. The pinch valves closethe fluid pathway by depressing the walls of the tubing against oneanother. It will be appreciated by those skilled in the art that suchpinch valves will be selected to accommodate the size of the tubingchosen for use as a fluid pathway. In addition, the compression force ofthe pinch valve will be selected to achieve the compression of thechosen tubing and thereby affect the closure of the fluid pathway. Thevalve specifications, therefore, will be matched to the softness orhardness (durometer) specifications of the selected tubing.

An embodiment of the separation device further includes recirculationloop 92 (fluid pathway seven) such that fluid that has already passedthrough separation column 40 may be recirculated through the separationcolumn. Typically, a pump 64 will be connected to the recirculation loopto facilitate the recycling of fluid through the separation column aswell as control the flow through the column. It will be appreciated bythose skilled in the art that a variety of pumps may be used. Anexemplary pump is a peristaltic pump that can control the passage offluid through the recirculation loop in either direction and at variablespeeds. The separation device having a recirculation loop allowssequential separations on one column, by a process of binding andelution, followed by a second binding and elution. Sequentialseparations provide improved purity in the final target population.

FIG. 2 schematically depicts a preferred embodiment of a separationdevice. It will be appreciated, however, that a separation process maybe accomplished using the basic system components, i.e. the improvedmagnetic separator and collection containers.

It will be appreciated by those skilled in the art that therecirculation means and fluid flow pathways of the present invention arealso suitable for use in alternate separation systems. For example, therecirculation means may be advantageously used in a system wherein theseparation means involves centrifugal techniques, absorption columns orchemical means as alternatives to the magnetic or electromagneticseparation means.

In an exemplary cell separation process, the fluid pathways and columnsare primed by allowing a wash liquid to flow through all of the fluidpathways and columns, preferably at varying flow speeds and pressures.The wash liquid may contain materials such as a physiologicallyacceptable protein, such as human serum albumin (HSA), which inhibitscells from adhering to the interior surfaces of plastic devicecomponents. The wash liquid may also contain small quantities ofphysiologically acceptable surfactants or detergents, to improve thewetting of the interior surfaces.

It is found that large air bubbles in the fluid pathways are detrimentalto the recovery of viable cells. Methods of removing air bubbles fromfluid pathways are known in the art, and may be employed for thispurpose. A method of particular interest exploits the observation that apermeable frit such as those used in the cap layer and/or base layerwill not allow passage of air bubbles at fluid flow rates of less thanabout 400 ml/minute. A column may be cleared of air bubbles bycirculating wash liquid through a recirculation loop at flow speeds ofless than about 400 ml/minute. The air bubbles then accumulate outsideof the column at the base and/or cap layers. The direction of flow isthen reversed, and the bubbles are washed out of the system into asuitable waste bag. Preferably, the sequence is repeated to ensure theremoval of all bubbles. The existing bubbles may be intentionallyenlarged by the generation of negative pressure.

In such an exemplary process, magnetically-conjugated antibodies may beused to specifically target the desired cells in a mixed cellpopulation. The magnetic reagent is incubated with the mixed cellpopulation, then unbound particles are washed away by any convenientmeans, e.g. centrifugation, etc. When cell sterility is desired, theantibody incubations and washes may be performed in a closed containerprocess, where the antibodies and wash liquids are added to a sterilecontainer by means of a sterile syringe or similar device. In this way,contamination of the desired cells by air-borne microorganisms isminimized. In such a closed system, particularly where the container isa flexible bag, the mixing of cells and antibody may be improved byinjecting a small amount of sterile air, at a ratio of from about 0.5 to2 of air to liquid, into the container.

The incubated cell suspension, now containing magnetically labeledtarget cells, is passed through the separation device. The systemtransports the cells through a magnetic separator which is positionedwithin or is adjacent to a magnetic field. The source of the magneticfield may be a permanent magnet or an electromagnet. The separationcolumn is preferably constructed to include a ferromagnetic matrix ofstacked ferromagnetic spheres, as described above. Optionally, thesample is passed through a prefiltration column, also constructed asdescribed above, prior to passage through the separation column. If theseparation column contains a matrix comprised of other thanferromagnetic spheres, then the sample may be first passed through aprefiltration column that is substantially identical to that separationcolumn.

The magnetically labeled cells accumulate in the separation column inresponse to the magnetic field. The non-labeled cells and othersuspension components pass through the separation column and into anunlabeled sample container and/or waste container. The labeled orpurified cells may then be eluted from the separation column by eitherremoving the separation column from the magnetic field or removing themagnetic field from the separation column. A wash solution, such as abuffered liquid, is passed through the separation column to wash thelabeled cells from the separation column and into a product collectioncontainer. The collection container may be used for further processingof the target cells or cryopreservation of the target cells.

In preferred embodiments, the separation column is a high gradientmagnetic separation column constructed from ferromagnetic spheres, asdescribed above. The containers referred to herein are typically plasticbags, such as those used for the storage and delivery of intravenousfluids, but any suitable containers can be used. The containers will beselected for their necessary storage or collection volume, theircapacity for sterilization and their ability to be used in a closedsystem, i.e. a separation system from which substantially all of the aircan be removed prior to use.

In another embodiment, the magnetically labeled cells are recirculatedthrough the separation column to enhance the selection of the targetcells and the removal of unwanted cells or other suspension components.Some preferred embodiments also include the use of a prefiltrationcolumn. The prefiltration column, however, is not, subjected to amagnetic field. Instead, the preliminary passage of the cell suspensionthrough the prefiltration column results in the capture of suspensioncomponents or materials that otherwise might non-specifically bind tothe separation column. Such non-specific binding may cause the blockageor fouling of the separation column, which could in turn inhibit orreduce the separation and collection of the labeled cell population.

The fluid pathways, collection containers, suspension filter,prefiltration column and separation column, may be constructed,interconnected and supplied as a disposable separation device. Thetarget cells are preferably collected in a sterile blood transfercontainer from which the cells can be transplanted to the patient or inwhich the cells can be stored or subjected to further processing. Thecomplete cell separation device may be pre-packaged in suitablecontainers. The pre-packaged device can be sterilized and provided readyfor use in the improved magnetic separation process of the presentinvention. The reagents necessary for the desired separation process mayalso be provided in kit form. For example, a conjugate specific for thetarget cell population or other analyte may be provided separately from,or with the device. The kit may also include wash solutions, for examplestandard sterile saline solution, and/or other buffered liquids, such asphosphate buffered saline, 1 mmol/l EDTA and 0.5% human serum albumin.These reagents or other solutions can be provided in containers such asplastic bags that can be connected to the appropriate fluid passages ofthe cell separation device.

The improved separation system of the present invention may be totallyautomated. In the automated system, a computer controls the flow offluids through the fluid circuitry and separation column, controls themagnetic field strength or placement of the magnet and/or separationcolumn to provide for the retention and release of the magneticallylabeled target cells or analyte, and directs the final collectionproducts into appropriate containers.

One embodiment of an automated cell separation instrument, as depictedin FIG. 3, includes mechanical, electromechanical and magneticcomponents. The mechanical components may include: an instrument outershell or housing 15; adjustable container holder 21; peristaltic pump64; prefiltration column holder 32, and separation column holder 42. Theelectromechanical components may include: solenoid pinch valves 1-11; aninternal motor (not shown) to drive peristaltic pump 64; an internalmotor (not shown) to move separation column holder 42 (and thereby movethe separation column 40) into or out of the magnetic field, or to movethe magnet 50; and a bubble detector (ultrasonic sensor) 65, which isused to detect the presence of fluid in the fluid circuitry. Themagnetic means may include permanent and electro-magnets. It will beappreciated that these individual components may be selected from anumber of readily available alternates and may be combined in a varietyof configurations without departing from the general description of theimproved magnetic separation system of the present invention.

In a preferred embodiment of the separation device, the fluid pathways,solution and collection containers, suspension filter, prefiltrationcolumn, separation column and connectors are provided as a preassembleddisposable component to the separation system. The separation device ismounted on the separation instrument for the performance of theseparation process. Upon completion of the separation process theproduct collection container may be removed, and the remainingseparation device components are disposed.

Preferably, an onboard microprocessor (not shown) controls all of theelectromechanical components of the instrument, and software directs thesystem to perform the appropriate operations in a standard sequence. Adisplay 62 and operator keypad 60 allow the operator to monitorautomatic system operation and to control instrument operation in amanual mode. A printer (not shown) may be connected to themicroprocessor for printing process information, labels, etc.

FIG. 3 depicts a separation instrument and mounted separation device. Inthis embodiment, the separation device is mounted or installed upon theinstrument by positioning the tubing of the fluid pathways in theirrespective external pinch valves 1-11, as described above. Prefiltrationcolumn 30 is placed within prefiltration column holder 32. Separationcolumn 40 is placed within separation column holder 42 which is movedrelative to magnet 50 by means of retractable arm 44. Adjustable clamp20 is used to secure hanger arm 21 in a raised position. There aremounts or pegs 22 on the hanger arm on which to place initial wash wastecontainer 82 and buffer and sample containers (not shown) asappropriate. The apparatus may further include storage compartment 70 toseparate the final waste container and unlabeled sample container fromthe product collection container (not shown).

As previously mentioned, separation systems as described above canfurther be integrated with various sample/cell processing systems foraccomplishing sample preparation steps prior to sample componentseparation by the described magnetic based separation techniques. FIG. 5illustrates a system according to an embodiment of the presentinvention. As described above, a system of the present invention caninclude various mechanical, electromechanical, and magnetic components.The system 100 includes a separation unit 106 and processing unit 104integrated into a single system containing an outer shell or housing105. The system 100 can include a magnetic separation system or unitsimilar to those described above. The system 100 includes a magneticseparation unit 106 including a housing for positioning a separationcolumn (e.g., magnetic separation column as described above, FIG. 1) inthe magnetic separation unit 106. The system 100 further includes a pump108 and a plurality of fluid flow control means or valves, asillustrated by valve 110. It is noted that, while only valve 110 isspecifically identified by number, the system 100 as shown in FIG. 5illustrates a number of valves that are identifiable as having anillustrated structure identical to that of valve 110. It will be furtherrecognized that while each of the plurality of flow control means/valvesare identical in FIG. 5 for illustrative purposes, flow control meansaccording to the present invention can take a variety of embodiments andcan include one or more different types of means/valves in a singlesystem. Components of the system 100 (e.g., valves, pump, separationunit, etc.) can be coupled or connected by one or more flow paths so asto form a series of fluid pathways or fluid circuits similar to asdiscussed above. The system further includes a computer control systemor unit 112 providing monitoring and/or control of one or more aspectsof the system 100.

The computer system 112, as described above, can include one or moreinput and/or output devices, graphical displays, user interfaces and mayallow for manual and/or automated con-trol of system 100 operation andfunctions. The computer control system 112 can include a module orsystem to process information (e.g., flow information, etc.) within thesystem 100 and can include a wide variety of proprietary and/orcommercially available computers, components or electronics having oneor more processing structures and the like, with such sys-tems oftencomprising data processing hardware and/or software configured toimplement any one or a combination of method steps as described herein.Software will typically comprise machine readable code of programminginstructions embodied in a tangible media such as a memory, digital oroptical recording media, optical, electrical, or wireless telemetrysignals, or the like, and one or more of these structures may also beused to output or transmit data, signals, or information betweencomponents of the system in any of a wide variety of signal processingarchitectures.

The system can further include various supports, sensors, housings, etc.for various components that can be coupled with the present system toperform methods as described herein. The system 100 further include oneor more support structures 114 configured to hold and/or support variousfluids, reagents, samples fluid reservoirs, filters, and the like thatcan be utilized with the system 100 according to the present invention.Support structures can include various hook or hanger, or holder (e.g.,filter holder or housing) configurations and are not limited to anyparticular design. Fluids, buffers, reagents, etc. positioned on asupport 114 can be coupled to a fluid path or tubing, that can in turnbe connected to more or more components of the system 100. The system100 can include sensors for monitoring and/or further controlling fluidflow through the system. Sensors can include, for example, liquidsensors, which can include bubble detectors (ultrasonic detector),pressure sensors, and the like. Bubble detector 116 and pressure sensors118 are shown. A support 120 is show, which can be configured to hold afilter or volume reduction unit. Collection area 122 can supportcollection containers, reagents, etc.

The system 100 is further illustrated with reference to FIG. 6.Processing unit 104 can include a housing or cover 124, that can bemovable (e.g., removable) about one or more hinge. The cover 124 atleast partially defines a processing area 126 that can be temperaturecontrolled and coupled to temperature monitoring and control componentsthat may be housed within the housing 105 of the system 100. Theprocessing unit 104 includes a sample chamber 128 configured for holdingand processing (e.g., centrifugation, culturing, sample componentseparation, etc.) of a sample. The sample chamber 128 shown is arotating chamber held in position about an axis that can include ananti-rotation lock 130. The processing unit 104 can include one or moredetection systems, such as an optical detector 132 positioned within thecover 124 and configured to detect or monitor processing of a sample inthe chamber 128. One or more fluid input/output lines can be coupled tothe chamber 128 and may be held in position by a holder 134.

FIG. 7 illustrates a backside view of the system 100. The computercontrol unit 112 is shown with a component coupled to the system housing105 about a rotational pivot, and the unit 112 having a storage media(e.g., program card) input slot 138. Hanger 140 is shown, which canprovide support for external components, collecting equipment, etc. Thesystem 100 includes power connect and switch 142 and various interfaceconnects 144 (e.g., barcode reader connect, printer connect, networkconnect, etc.); vent 146; and heat sink 148 providing a component ofinternal temperature control systems.

Components of a processing unit, including a sample chamber, are furtherdescribed with reference to FIGS. 8 through 12. Referring to FIG. 8,processing chamber 150 is illustrated having an upper portion 152 and alower base portion 154. The upper portion 152 can include areinforcement or support structure 156. The chamber 150 further includesan axis 158 about which the chamber 150 rotates, the axis 158 having arotational lock, and the axis 158 extending through about the center ofthe chamber 150 and extending out the upper portion 152. Rotationalmeans or bearing 160 provides rotational movement of the chamber 150about the axis 158. The chamber 150 further includes fluid ports or lineconnects 162, 164 coupled to a housing structure surrounding the axis158, and ports 162, 164 fluidly connected to one or more internalcompartments of the processing chamber 150.

One of the ports may be used as vent to exchange gases.

A processing chamber according to another embodiment of the presentinvention is described with reference to FIG. 9. The chamber 170includes an upper portion 172 and a lower portion 174, with a rotationalaxis 176 and ports 178, 180 configured similarly as described above. Theupper portion 172 includes a support structure 182 as well as structure184 including a channel 186 that can include at least a portion visiblethrough a window or prism 188.

The channel 186 can be fluidly coupled to a sample containmentcompartment in the chamber 170 and configured for external monitoring ordetection of sample processing. For example, a component (e.g., cells)in fluid in the channel 186 may become visibly separated duringprocessing steps, thereby indicating separation of cells or samplecomponents in one or more internal compartments of the chamber.

The chamber 170 may further comprise at least one vent, preferentiallycomprising a sterile, hydrophobic membrane or tampon. Preferably thesemembranes or tampons may be located at the top or bottom of the chamber.The at least one vent in the chamber has the particular ad-vantage thatthe volume in the chamber can be changed easily without changing thepressure in the chamber or providing further inlet an/or outlet portsfor the exchange of air or gas.

The centrifuges of the present invention permit a batch-wise as well asa continuous centrifugation: sample, media, gases and other materialscan enter and leave the system e.g. through inlet and outlet ports (e.g.178 and 180 in FIG. 1) without a need of stopping the rotation of thecentrifugation chamber and refilling the centrifuge (batch-wisecentrifugation). This allows a continuous concentration of the sampleand the product may be removed only once at the end of thecentrifugation thus avoiding potential contamination due to additionalhandling.

In FIG. 9 a, a rotating container or centrifugation chamber 500 isshown. At the bottom of the rotating chamber 500 a microscope focus area505 is positioned which comprises at least one sensor pad 504. Below therotating chamber 500, a microscope camera module 503 is located thatcomprises a microscope optics 501 and a microscope drive motor 502 forfocusing the optics. The microscope optics 501 is configured such thatis can focus automatically to detect the sample that is being separatedinto at least two components during centrifugation. Thereby, themicroscope camera module 503 can be used to detect different layersformed by the separated sample in the chamber 500 due to centrifugalforces. In addition, the pH value of the sample components can bemeasured. For this purpose, an indicator is used in the chamber 500 thatchanges its color dependent on the pH value that is present. Moreover,it is possible that the temperature of the sample in the chamber bemeasured using liquid crystals that are position in the chamber suchthat their position can be detected with a microscope camera module 503from the outside. Thereby, the temperature in the chamber 500 can bedetermined.

The microscope camera module 503 can be mounted in a movable fashion,such that the module 503 can be directed with its microscope optics 501at different sensor pads 504 located in the wall of the chamber 500.This facilitates the detection of various layers formed in the chamber500 or the detection of the pH or the temperature at different positionswithin the chamber 500.

FIG. 10 illustrates a cross-sectional view of a sample processingchamber according to an embodiment of the present invention. The chamber190 includes an upper portion 192 and a base portion 194, and one ormore internal compartments. The chamber 190 is configured to rotateabout an axis so as to apply a centrifugal force to a sample disposed inone or more compartments in the chamber, thereby separating at least twocomponents of the sample. The chamber includes central line 196 fluidlyconnected to at least one compartment of the chamber. Components of thechamber 190 further include outer line 198; rotational bearing 200,rotational seals 202, 204, 206; outer entry line to the chamber 205;lower radial channel 208; inner line entry 210 to a chamber compartment;slant 212, and deflector 214. Chamber retainer 216 is included andconfigured for secure positioning/coupling of the chamber 190 with othercomponents of a system of the invention.

The centrifugation chamber 190 preferably comprises a rotating seal,optionally with two fluid lines, preferably with two fluid lines. Thefluid lines can enter the chamber 190 at different position. Forexample, it is possible to position a first fluid line at the outerperimeter of the upper portion 192 (lid). A second fluid line could bepositioned further inward, e.g. 2 mm to 20 mm further towards the centerof the chamber 190. Optionally, a vent can be located at the upperportion 192, e.g. in the form of a membrane.

Generally, the position of openings such as holes or line entries in thecentrifugation chamber can be configured such that they are best suitedfor the centrifugation of a particular sample. Depending on thecomponents of a particular sample, and the relative volume of eachcomponent in the sample, the openings can be positioned so that theremoval and/or detection of a particular component can be achieved.

FIG. 10A illustrates a top plan view of a chamber 201. The chamber 201includes an inner line 203, lower radial channel 205, inner line entry207 to the chamber, optionally a deflector 209, a slant 211 and a lightpass 209.

FIG. 11 illustrates a cross-sectional view of a chamber according toanother embodiment of the present invention. The chamber 220 includes anaxis about which the chamber rotates, a central line connect 222 and aouter line connect 224, and one or more internal compartments. Furtherillustrated are rotational bearing 226, as well as rotational seals 228,230, 232; inner channel 234, optical detection channel 236 (similar todescribed above); inner line entry 238 to the chamber; inner line 240,and lower radial channel 242. The chamber further includes an innerreinforcement 246 and a chamber retainer 248. FIG. 11A illustrates afocused view of portion of a chamber 220 is described above. Shown arean optical detection channel 236, a prism 237, and light pass 239(further indicated by arrows).

In another embodiment of the present invention, the bottom of thechamber can possess one or more openings (FIG. 12A, 291) covered with ahydrophobic membrane 292. These openings can be used for gases to bedelivery into or removed from the chamber, for instance for cell cultureprocesses. The membrane can be glued, thermally, ultrasonic or by othermeans bonded to the bottom of the chamber in a way to assure sterileconnection with the chamber.

In another embodiment of the present invention (FIG. 12B), the chambercan possess a system of channels for the gas flow, for instance channelsassembled as a spiral system 293, which assures a large contact areabetween the gases and a membrane bonded over the channels (not shown).These channels may be located at the bottom or the top of the chamber.The channel system possesses at least one input (opening) 294 and anoptional output (opening) 295 for the gases.

The entries or ports of the channels of FIGS. 8-11A may vary in numberand location within the channel.

FIG. 12 illustrates a cross-sectional view of a chamber according toanother embodiment of the present invention. Construction of chamber 250is similar in many regards to chambers as described above, but furtherincludes a plurality of layered structures 252. The layered structures252 can be configured to provide cell culture structures or layers. Inuse, sample including cells can be introduced into the chamber andflowed over layers 252. Separation processing can include rotation ofthe chamber such that cells adhering to the layers are separated fromthose with lesser affinity for the layers. Intermittent rotation and/orbreaking during rotation can further disconnect cultured cells from thesurface of the layered structures 252 for separation processing.Surprisingly, it was found that this intermittent process is also ableto resuspend clumped or attached biological material, like cells afterculturing. The chamber further includes illustrated central line 251,outer line 253, bearing 255, rotational seals 257, outer line entry 259to the chamber, upper portion 261, inner channel 263, base portion 265,retainer 267, lower radial channel 269, and inner line entry 271 to thechamber.

The chamber may comprise or may be made of various materials. In apreferred embodiment, transparent materials are used like plastics,polystyrol (PS), polysterene, polyvinylchloride, polycarbonate, glass,polyacrylate, polyacrylamide, polymethylmethacrylate (PMMA), and/orpolyethylenterephtala (PET). Polytetrafluorethylen (PTFE) and/orthermoplastic polyurethane (TPU), silicone or compositions comprisingone or more of the above mentioned materials. The chamber can also bemade of polyethylene (PE). In a preferred embodiment, the layers in thechamber comprise or are made of collagen, chitin, alginate, and/orhyaluronic acid derivatives. Possible are also polyactide (PLA),olyglycolida (PGA) and their copolymers, which axe biodegradable.Alternatively, non-biodegradable materials can be used, such aspolystyrol (PS), polysterene, polycarbonate, polyacrylate, polyethylene(PE), polymethylmethacrylate (PMMA), and/or polyethylenterephtala (PET).Polytetrafluorethylen (PTFE) and/or thermoplastic polyurethane (TPU) canalso be used. Other alternatives include ceramics and glass materials,like hydroxylapatite (HA) or calcium phosphate. The layers in thechamber can be of solid material or porous.

In a preferred embodiment the chamber has a size of 2 cm to 50 cm indiameter and a height of 5 mm to 50 cm. Centrifugation is preferentiallycarried out up to 1000×g.

The number of the layers and the distance between the layers isvariable.

In a preferred embodiment, the chamber can be heated and cooled toprovide for a temperature appropriate for the sample to be centrifuged.For this purpose, a heating and/or cooling means is located in thesystem.

As shown in FIG. 17, the cylindrical shaped centrifuge chamber my belimited on its upper side by a lid 800, which may carry one or morestabilizing ribs 805 on the flat top surface. At least one of theseradial ribs 805 can cover a narrow gap or channel 801, open to thecentrifuge's inner volume when the lid 800 is tied on the centrifugechamber. The gap 801 extends in axial direction from the inner lidsurface passing the lid 800 some millimeters into the rib 805.Therefore, it may be visible from the outside within the rib 805 whentransparent material is used. In radial extension, the gap 801 reachesfrom near the center up to the cylindrical centrifuge wall (FIG. 17).

During centrifugation, the same forces take effect in the gap 801 as inthe whole centrifuge chamber. The ring shaped neighbored suspensionlayers extend parallel into the gap 801 and are displayed as axialstanding neighbored thin areas, like a thin layers cross cut, welldetect-ible by external optical sensors.

The gap 801 width can be determined freely, also small enough for atransmitted light analysis of all layer-associated areas in the gap.Thereby, it is possible to quantify the optical densities and colors ofall layers of the suspension in the centrifuge chamber in a “touchless”manner from the outside through optical transmission measurements.

To enable a vertical illumination and sensor position to watch thelayers movements in the gap, a window, a mirror or preferentially aprism can be added on both rib sides, which may preferably be preformedby the transparent housing material itself.

The prism 810 refracts the vertical generated illuminating beam throughthe gap (horizontal) and back to the top, vertical again (FIG. 18).During centrifugation, a synchronous position triggered electronic flashlight can transmit light into one side of the prism 810, e.g. the leftprism, illuminating the gap by refraction. The transmission result isrefracted by the other side of the prism 810, e.g. the right prism, backto a vertical mounted sensor or camera, possibly in the neighborhood tothe upper flash source on the top. The resulting optical sensor unit iseasy to handle like a reflex sensor but at the same time allows forfull-scale transmission measurements.

The arrangement of the prism's angels ensures the “total reflection” onits inner prism surface for the illuminating flash beams and avoidsdirect reflections on its outer surfaces between light source andcamera. Therefore, there is no need for mirror coatings and injectionmolding technologies can be used without rework of the facilities beingrequired (FIG. 19).

Another embodiment of the present invention is described with referenceto FIG. 13. As illustrated, a processing system including variouscoupled components, flow channels, buffers, reagents, etc. It will berecognized that numerous configurations are available and that thecurrent configuration is provided for illustrative purposes. Referringto FIG. 13, components include a system buffer 300, spike port 301,sterile filter 302, plasma/in process bag 303, magnetic labeling reagentcontainer 304, spike port 305, magnetic reagent sterile filter 306,sterile filter 307, buffer/media bag 308, cell culture media port,auxiliary port 309, single direction valve downwards 310 possiblyincluding a filter, single direction valve upwards 311, sample bag 312,sample bag connector 313, sample filter 314, sample port 315, filter316, pre-separation filter 320, in process storage bag 321, magneticseparation column 322, waste bag 323, volume reduction unit 324,positive fraction bag 325, negative fraction bag 326, sterile air filter327, pump 328, air filter to pressure sensor1 329, air filter topressure sensor2 330, sample/cell processing unit 332.

A typical use of a system as illustrated in FIG. 13 and described above,is discussed with reference to FIGS. 14A through 14N. Activation ofvarious components of the system can be selected to direct flow along adesired path. Selected flow is illustrated with reference to FIGS. 14Athrough 14N and shown by darkened flow paths and flow direction arrows.FIG. 14A illustrates sample loading into a rotating/processing chamberof the sample processing unit. Sample flows from the sample sourcethrough open valves 5, 9, 16 and through the pump and into theprocessing unit. FIG. 14B illustrate rinsing of lines/components of thesystem, with buffer flowing through open valves 1, 9, 16, pump, and intothe processing unit. Sample and buffer can be flowed as illustrated,sample in the processing unit centrifuged for separation of components,and the rinsing process repeated. FIG. 14C illustrates red blood cellreduction and removal from the sample. As stated above, sample in theprocessing unit can be centrifuged for separation of components,including formation of a buffy coat for separation of red blood cells.Red blood cell component can then be removed from the processing unitand flowed through the system through open valves 17, 12 and into thewaste container. FIG. 14D illustrates removal of plasma from the chamberin the processing unit, where plasma rinse/component is flowed throughthe system through open valves 16, 12 and into the waste container. FIG.14E illustrates collection of plasma as plasma is removed from theprocessing unit, flowed through the system at open valves 16, 9, 4 andinto a plasma collection container. FIG. 14F illustrates loading ofreagent into the chamber and labeling of cells. Reagent (e.g., magneticlabel) is flowed through the system at open valves 2, 9, 16 and into thechamber. Cells can be processed (e.g., agitated, reconstituted, mixedwith reagent, etc.) for incubation with label reagent. FIG. 14Gillustrates addition of plasma into the chamber. Plasma from the plasmacontainer is flowed through the system through open valves 4, 9, 16 andinto the chamber. Additional processing, rinsing, etc. steps (e.g., asillustrated in FIG. 14B) can be preformed. FIG. 14H illustrates magneticbased separation of labeled and unlabeled components of the sample.Sample/resuspended labeled cells are flowed through the system throughopen valves 16, through a filter or (pre-)column (e.g., size basedseparation filter so as to remove clumped cells), open valves 7, 11,through the magnetic column where labeled cells are bound, withprimarily unlabeled cells flowing through open valve 19 and intonegative/unlabeled cell container. FIG. 14I illustrates column rinsingwhere buffer is flowed through the system at open valves 1, 15, 7, 11,19 and into the cell container. Rinsing steps are selected to washunlabeled cells from the system/magnetic separation column. FIG. 14Jillustrates elution of cells from the column and reapplication. Magneticapplication is turned off and fluid flowed as illustrated through openvalves 11, 10, 14, e.g., at high speeds, so as to wash cells from thecolumn. The magnetic field application is then turned back on forreat-tachment of labeled cells to the magnetic separation column. FIG.14K illustrates rinsing of the column with an elution buffer. Buffer isflowed as illustrated so as to preferentially wash unlabeled cells fromthe separation column and into the waste container. FIG. 14L illustratesflushing of the column so as to remove labeled cells from the column andinto the reapplication bag. As shown, the magnetic field is turned offto free labeled cells from the column. FIG. 14M illustrates volumereduction steps. Solution containing cells is flowed as illustrated soas to pass the solution through the filter F7. The filter includes amembrane filter that collects cells as solution flows through. FIG. 14Nillustrates final elution and collection of the product into thecollection container. Plasma is flowed as illustrated through openvalves 4, 9, 14, 18, with fluid passing through filter F7 so as to washcells from the filter and into the positive collection container.

The tubing set of the present invention can be used for delivery ofnutrient medium, cytokines, growth factors, serum and other substancesnecessary for the cultivation of the cells, or for taking out samplesfrom the cultivation chamber. Nutrient medium can be continually orperiodically delivered into the chamber or withdrawn from the chamberthrough the ports 222 and 224 (FIG. 11) by using the pump 328 (FIG. 13)or by using additional syringes connected to the tubing set. The mediumcan be completely or partially exchanged with fresh medium during thecell cultivation process. The medium can be enriched with O₂, CO₂, N₂,air or other gases necessary for the growth of the cells. The mediumenrichment can be done by direct injection of the gases into thecultivating chamber through the ports 222 or 224 (FIG. 11), through anadditional opening at the bottom of the cultivation chamber covered witha hydrophobic membrane (FIGS. 12A and 12B) and/or by using an aerationdevice, positioned outside the cultivation chamber (FIG. 15).

An example of such an aeration device is depicted in FIG. 15. Itconsists of two parts (415 and 416) made of, for instance, polycarbonate(PC) or polymethymetacrylate (PMMA) with en-graved channels on one side.Both parts are brought together forming a hollow space in between,separated in two or more compartments by at least one membrane (417).The membrane can be bonded to one of the parts or several membranes canbe bonded on both parts. The compartments contain at least one inlet(418) and one outlet (419) port for connection with the tubing set. Thefirst compartment is used for the gas-mixture. The second compartment isused for the nutrient medium, which has to be enriched with thecomponents of the gas-mixture. The membrane positioned between bothcompartments is permeable for gases and not permeable for liquids.Preferentially, this is one hydrophobic membrane, for instance nylonhydrophobic membrane. Preferentially, the pores of the hydrophobicmembrane are smaller then 0.2 μm, which assures a sterile connectionbetween the nutrient medium and the gas-mixture. In another embodimentof the aeration device, one or more additional membranes can be bondedto either of the parts depicted in FIG. 15, in a way that the gas flowsbetween the medium containing compartments. In this case, the contactsurface between the medium and the gas-mixture is larger by multitudesdepending on the number of membranes used.

The membranes in the aeration device can be bonded to the parts by meansof thermal bonding, ultrasonic bonding or gluing, or other suitablebonding process, which allows a sterile connection between the plasticparts and the membrane.

The aeration device can be sterilized by means of irradiation, (e.g.gamma-, beta-radiation), plasma- (e.g. hydrogen peroxide), hotvapor/steam (e.g. autoclaving) or ethylene oxide (EtO) sterilization.Preferentially the aeration device is used as a part of the disposabletubing set.

The gas-composition used for the aeration device is preferentiallycomposed through use of a gas-mixing chamber. A preferred embodiment ofthe gas-mixing chamber is shown in FIG. 16 A. It consists of two parts(421 and 422), preferably made of plastic material, for instance POM(Polyoxymethylene), which are connected with bolts, glued, thermally orultrasonic bonded together in a way to form a hollow space between them.Preferentially, one of the parts contains a channel for a sealing ring,which assures the sealing between both parts (433, FIG. 16B). Thegas-mixing chamber possesses at least one inlet port and at least oneoutlet port (423). It comprises a port (430) for connection of apressure sensor device for measurement of the pressure inside thechamber. The gas-mixing chamber optionally contains another port (432,FIG. 16 b) for connection with a safety valve, which opensautomatically, when the pressure in the chamber rises over a certainvalue. The gas-mixing chamber possesses inlets and outlets (435) forconnection with at least one inlet and at least one outlet valve, aswell as drillings with threads (436) for connection of the inlet andoutlet valves to the walls of the gas-mixing chamber.

For reduction of the flow rates of the gasses into and/or out of thegas-mixing chamber, blends can optionally be included in the walls.

Principle of operation of the gas-mixing chamber: the inlet ports of thegas-mixing chamber are connected with a gas supply. The supply of theinlet gasses should have pressure of more than the atmospheric pressure,e.g. 2 bar. The operator must give the content of the desired gascomposition by using a measurement and automation software program. Thecontent of each of the gases of the desired composition is given as thepartial pressure of that gas in the gas mixture. The gas compositionprocess starts with the first component of the gas-mixture, which iscontrolled by the input valve 424 (FIG. 16A). The input valve opens fora period of time, usually between 50 ms and 200 ms, and then closes andthe pressure sensor is read out. The measured pressure in the chamber isP_(m). If the pressure P_(m)<P₁, where P₁ is the pressure given by theoperator, the input valve opens again. The process is repeated untilP_(m)>=P₁. Then input valve 425 opens and the composition of the secondgas component takes place as described in the case with input valve 424.When all input gases are composed into the mixing chamber, the outputvalve 427 opens until the pressure in the gas-mixing chamber reaches avalue P_(out), preliminary given by the operator. P_(out) is alwayseither equal to the atmospheric pressure or higher. The time and thefrequency of the opening of the outlet valve can be given by theoperator as well. Then the gas-composition process starts again with theopening of the first inlet valve (424). Outlet valves 428 and 429 can beoptionally used for aeration of another cell culture chambers, bags orother vessels used in the present invention.

The sample chamber allows a large range of cell culture methods to beperformed, such as growing of cells, separating, washing, enriching thecells or different kinds of cells, or others. The system of theinvention can also be used to formulate drugs.

Manufacturing of cell preparations required for cellular therapy caninclude various combinations of basic operations to obtain a definedcell product with defined characteristics. It is unique to the inventionthat all these basic operations can be performed in a single, closedtubing set, eliminating the risks of otherwise required manual sampletransfer steps.

Cell culture conditions for the examples are known in the art.

The features described herein may be of relevance for the realization ofthe present invention in any combination.

EXAMPLES

The following examples are provided to illustrate, but not limit theinvention.

Example 1 Manufacturing of Stem Cell and Progenitor Grafts from CordBlood

Human cord blood is diluted with CliniMACS PBS/EDTA Buffer, adjusted tothe defined labeling volume by centrifugation, CliniMACS CD34 or CD133Reagent is added and allowed to incubate with the cell sample for thespecified labeling time, access reagent is removed by washing in thecentrifuge chamber, CD34 or CD133 positive cells are enriched from thecell suspension and eluted from the MACS column directly with cellculture medium and transferred into the cell culture chamber. Mediasupplements (human serum albumin, cytokines) are automatically addedfrom the vial attached to the tubing set. Enriched or isolated stemcells are cultivated for up to three weeks. Supplemented cell culturemedium is intermittently added. Expanded cord blood cells are washed bycentrifugation to remove cell culture medium and cells are resuspendedin infusion solution (saline) supplemented with human serum albumin andtransferred to the final product container (infusion bag or syringe).

Example 2 Manufacturing of a Dendritic Cell Vaccine

Human whole blood or apheresis harvest is diluted with CliniMACSPBS/EDTA Buffer, adjusted to the defined labeling volume bycentrifugation, CliniMACS CD14 Reagent is added and allowed to incubatewith the cell sample for the specified labeling time, access reagent isremoved by washing in the centrifuge chamber, CD14 positive monocytesare enriched from the cell suspension and eluted from the MACS columndirectly with cell culture medium and transferred into the cell culturechamber. Media supplements (human serum albumin, cytokines) areautomatically added from the vial attached to the tubing set. Enrichedor isolated monocytes are cultivated into immature monocyte-deriveddendritic cells. Media is exchanged by centrifugation and additionalsupplements are automatically added from the vial attached to the tubingset. Cells are cultivated and upon maturation antigens (recombinantprotein, peptides, cell lysates, DNA) are automatically added from thevial attached to the tubing set. Monocyte derived dendritic cells arecultivated for antigen processing. Cell culture medium is removed bycentrifugation and cells are resuspended in infusion solution (saline)supplemented with human serum albumine and transferred to the finalproduct container (infusion bag or syringe).

Example 3 Manufacturing of a mDC and pDC Blood Dendritic Cell Vaccine

Apheresis harvest is diluted with CliniMACS PBS/EDTA Bufferautomatically depleted of CD19 and subsequently enriched for CD304(BDCA-4, CD1c, Neuropilin-1) or CD1c (BDCA-1) via the MACS column. DCsare eluted from the MACS column directly with cell culture medium andtransferred into the cell culture chamber. Media supplements (humanserum albumin, cytokines, activating components like recombinantprotein, peptides, cell lysates, DNA) are automatically added from thevial attached to the tubing set and the cells are cultured for 24 h toachieve an optimal maturation and activation. Cell culture medium isremoved by centrifugation and cells are resuspended in infusion solution(saline) optionally supplemented with human serum albumine andtransferred to the final product container (infusion bag or syringe).The product container is removed from the tubing set of the inventionfor further user.

Example 4 Manufacturing of Antigen Specific T Cells

Human whole blood or apheresis harvest is washed and diluted with cellculture medium and transferred into the cell culture chamber. Antigen(recombinant protein, peptide pool or tumor cell lysate) and mediasupplements (human serum albumin, cytokines) are automatically addedfrom the vials attached to the tubing set. Cells and antigen arecultivated for 3-16 hours to re-stimulate antigen specific T cells. Cellsuspension is adjusted to the defined labeling volume by centrifugation,CliniMACS IFN-gamma Catchmatrix Reagent is added and allowed to incubatewith the cell sample for the specified labeling time, and access reagentis removed by washing in the centrifuge chamber. Cells are transferredinto the cell culture container and incubated for release of cytokines.Cell suspension is adjusted to the defined labeling volume bycentrifugation, CliniMACS IFN-gamma Enrichment Reagent is added andallowed to incubate with the cell sample for the specified labelingtime, access reagent is removed by washing in the centrifuge chamber,antigen specific cells are magnetically enriched from the cellsuspension, and concentrated to a small, injectable volume by directelution from the MACS column using supplemented infusion solution andtransferred to the final product container (infusion bag or syringe).

Example 5 Manufacturing of a Activated Natural Killer Cells

Human whole blood or apheresis harvest is diluted with CliniMACSPBS/EDTA Buffer, adjusted to the defined labeling volume bycentrifugation, CliniMACS CD3 Reagent is added and allowed to incubatewith the cell sample for the specified labeling time, access reagent isremoved by washing in the centrifuge chamber, CD3 positive cells aredepleted from the cell suspension. CD3 negative target cells areadjusted to the labeling volume, CliniMACS CD56 Reagent is added andallowed to incubate with the cell sample for the specified labelingtime, access reagent is removed by washing in the centrifuge chamber,CD56 positive cells are enriched and eluted from the MACS columndirectly with cell culture medium and transferred into the cell culturechamber. Media supplements (human serum albumin, cytokines such as IL-2and/or IL-15) are automatically added from the vial attached to thetubing set. Enriched or isolated NK cells are cultured for 8-48 hours.Cell culture medium is removed from the cell product by centrifugationand cells are resuspended in infusion solution (saline) supplementedwith human serum albumin and transferred to the final product container(infusion bag or syringe).

Example 6 Manufacturing of Expanded Natural Killer Cells

Human whole blood or apheresis harvest is diluted with CliniMACSPBS/EDTA Buffer, adjusted to the defined labeling volume bycentrifugation, CliniMACS CD3 Reagent is added and allowed to incubatewith the cell sample for the specified labeling time, access reagent isremoved by washing in the centrifuge chamber, CD3 positive cells aredepleted from the cell suspension. CD3 negative target cells areadjusted to the labeling volume, CliniMACS CD56 Reagent is added andallowed to incubate with the cell sample for the specified labelingtime, access reagent is removed by washing in the centrifuge chamber,CD56 positive cells are enriched and eluted from the MACS columndirectly with cell culture medium and transferred into the cell culturechamber. Media supplements (human serum albumin, cytokines) areautomatically added from the vial attached to the tubing set. CellExpansion Beads loaded with antibodies directed against CD2 and CD335(NKp46) or CD314 (NKG2D) and CD335 (NKp46) are transferred from the vialto the cell culture compartment. Enriched or isolated NK cells arecultured for one weak, starting at day 7, supplemented cell culturemedium is added every 3 days in a 1:1 ratio and cells are cultivateduntil day 14-21.

Expanded NK cells are passed over the MACS column to remove cellexpansion beads. Optionally expanded NK cells are further purified byCD3 depletion or CD56 enrichment (see above). Cell culture medium isremoved from the cell product by centrifugation and cells areresuspended in infusion solution (saline) supplemented with human serumalbumine and transferred to the final product container (infusion bag orsyringe).

Example 7 Manufacturing of a Expanded T Helper Cells

Human whole blood or apheresis harvest is diluted with CliniMACSPBS/EDTA Buffer, adjusted to the defined labeling volume bycentrifugation, CliniMACS CD4 Reagent is added and allowed to incubatewith the cell sample for the specified labeling time, access reagent isremoved by washing in the centrifuge chamber, CD4 positive cells areenriched and eluted from the MACS column directly with cell culturemedium and transferred into the cell culture chamber. Media supplements(human serum albumin, cytokines) are automatically added from the vialattached to the tubing set. Cell Expansion Beads loaded with antibodiesdirected against CD2, CD3 and CD28 or alternatively CD3 and CD28 aretransferred from the vial to the cell culture compartment. Isolated Thelper cells are cultured, starting at day 3, supplemented cell culturemedium is added every 2 days in a 1:1 ratio and cells are cultivateduntil day 14. Expanded T cells are passed over the MACS column to removecell expansion beads. Cell culture medium is removed from the cellproduct by centrifugation and cells are resuspended in infusion solution(saline) optionally supplemented with human serum albumine andtransferred to the final product container (infusion bag or syringe).The product container is removed from the tubing set of the inventionfor further user.

Example 10 Selection of CD133 Positive Stem Cells

The CD133 antigen is a stem cell marker and is specifically expressed onan immature subset of CD34⁺ cells, on circulating endothelial progenitorcells and on a CD34⁻ stem cell subset (De Wynter et al., 1998). Enrichedor isolated CD133 positive cells using the CliniMACS CD133 System havebeen used for ex vivo expansion of hematopoietic progenitor cells formcord blood, and in autologous (Pasino et al., 2000) and allogeneic (Köhlet al., 2002) transplantation.

CD133⁺ stem cells can be utilized in non-hematological applications forregenerative medi-cine. CD133 selected cells from bone marrow have comeinto focus for example regarding the treatment of ischemic heartdiseases (Stamm et al., 2003).

Enrichment or isolation of CD133+ cells results in a removal of nontarget cells of more than 99.4% (2.2 log). See table 1.

TABLE 1 CD133 positive cells have been isolated from bone marrow usingthe present invention: Start Starting Final cell volume frequency Finalpurity CD133⁻ log number CD133⁺ [ml] CD133+ CD133+ depletion CD133+yield* 17 0.51% 56.54% 2.2 5.5E5 60.89%* 42 0.26% 51.15% 2.5 1.3E687.32%* 53 0.62% 66.16% 2.7 2.5E6 56.65% 34 0.26% 48.89% 2.3 8.4E577.96%* 62 0.55% 60.51% 2.7 1.8E6 54.04% (*calculated in relation toCD133⁺ cells recovered in all bags)

For comparison, all bone marrow products have also been processed usingthe CliniMACS® CD133 System with similar results.

The dotplots in FIG. 20 show sample characteristics before (left) andafter (right) automated processing using the present invention.

Example 11 Selection of CD14 Positive Cells

The CD14 antigen belongs to the LPS receptor complex and monocytesstrongly express the antigen. CD14 selected monocytes can be used forsubsequent generation of human mono-cyte-derived dendritic cells (MoDCs;Campbell et al., 2005). Dendritic Cells have great potential as cellularvaccines for various diseases, including solid tumors, hematologicalmalig-nancies, viral infections and autoimmune diseases.

Monocytes have been isolated from two leukapheresis harvests using thepresent invention and the CliniMACS CD14 Reagent. Monocytes wereenriched from 19.7%/31.8% (unprocessed harvest) to 98.2%/99.7% (finalcell product) with a yield of 58%/21% (see FIG. 21)

MoDC could be generated from monocytes isolated using the presentinvention and showed identical characteristics to MoDCs generated fromCliniMACS isolated monocytes.

Monocytes were also isolated from buffy coat from human whole bloodusing the present invention and could be enriched from 9.8% (unprocessedbuffy coat) to 98.9% purity (final cell product) with a monocyte yieldof 64%.

Example 12 CD304 Selection

In peripheral blood CD304 (BDCA-4, Neuropilin-1) is expressed on humanplasmacytoid dendritic cells (PDCs). PDCs are the most potent Type IInterferon-producing cells (Dzionek et al., 2002). CD304 can be used todirectly enrich or isolate dendritic cells from peripheral blood withouta culturing period (as for MoDCs).

Enrichment or isolation of CD304 positive target cells requires extendedwashing steps which are time-consuming utilizing the CliniMACS samplepreparation procedure in a blood bag centrifuge. Automated sampleprocessing will facilitate use of CD304 isolated cells in vacci-nationtrials.

CD304 positive cells were isolated or enriched from two leukapheresisharvest utilizing the present invention and CliniMACS CD304 Reagent.PDCs were enriched from 0.29% to 34.4% with a yield of 30% and from0.43% to 80.5% with a yield of 39%.

Plasmacytoid Dendritic Cells from the second procedure were cultivatedfor 24 h with CPG C (ODN2395) and IL-3 or IL-3 alone for maturationinduction. Cultivated cells were analyzed for maturation markers CD40and CD80 and marker profile was identical to isolated CD304 positivePDCs using the CliniMACS CD304 System upon cultivation.

Example 13 Selection of CD4 Positive Cells

CD4 is an accessory molecule in the recognition of foreign antigens inassociation with MHC class II antigens by T cells. T helper cells and toa lower degree monocytes and dendritic cells express CD4.

For certain oncological settings preliminary data suggest that T cellsin a graft or as a donor lymphocyte infusion may be beneficial(Falkenburg et al., 1999). In these settings a CD4 selection or CD8depletion (see example 5) may be desired in the allogeneictransplantation setting. T helper cells may be capable of mountinganti-tumor responses, reducing the risk of Graft versus Host Disease(Alyea et al. 1998) and reconstituting the recipient's immune system(Bellucci et al., 2002).

CD4 positive T helper cells also play a significant role in HIVinfection and progression. Selected T helper cells (potentially aftergenetic modification) may be desired for treatment of HIV complications.

CD4 positive cells were isolated or enriched from three buffy coatproducts from human whole blood utilizing the present invention and theCliniMACS CD4 Reagent. T helper cells were enriched from 27%/8.6%/15.9%to 90%/80%/81.3% purity with a yield of 93%/98%/51%. These results arewell within the range of results obtained using the CliniMACS CD4System.

Example 14 Depletion of CD8 Positive Cells

The CD8 antigen, a co-receptor for MHC class I molecules, is expressedon cytotoxic T cells and dimly on a subset of NK cells.

In the allogeneic transplantation setting CD8 T cells may be removedfrom a graft or donor lymphocyte infusion with the goal of preventingGraft versus Host Disease while maintaining Graft versus Leukemiaeffects Meyer et al. 2007).

CD8 positive cytotoxic T cells were depleted from three huffy coatproducts utilizing the present invention and the CliniMACS CD8 reagent.Cytotoxic T cells were depleted from 11.5%/8%/12% to0.005%/0.004%/0.002% respectively. This represents a >99.97% (>3.5 log)removal of non-target cells. CD8 negative target cells were nearlycompletely recovered (85.1%/90.9%/94.4%).

Example 15 Preparation of Peripheral Blood Mononuclear Cells by DensityGradient Centrifugation

Ficoll-Paque™ is a sterile density medium for the isolation ofmononuclear cells from bone marrow, peripheral blood, and cord blood. Itis an aqueous solution of density 1.077 g/ml, consisting of Ficoll PM400(a highly branched, high-mass, hydrophilic polysaccharide), so-diumdiatrizoate and disodium calcium EDTA. Erythrocytes and granulocyteshave a density of more than 1.077 g/ml while lymphocytes and monocyteshave a lower density. When human blood is layered on top of theFicoll-Paque lymphocytes and monocytes can be separated fromerythrocytes and granulocytes under centrifugal force.

The present invention has been used for preparation of peripheral bloodmononuclear cells by density gradient centrifugation. Buffy coat fromhuman whole blood was layered onto a ring of Ficoll-Paque while thecentrifugation chamber was rotating. The rotation speed had beenadjusted to 2,900 rounds per minute and maintained for 20 minutes. Anouter ring of erythrocytes and granulocytes, a ring of Ficoll-Paque, aring of target cells (peripheral blood mononuclear cells, i.e.lymphocytes and monocytes) and an inner ring of platelet containingblood plasma and PBS buffer could be detected. The different layers wereremoved from the centrifugation chamber of the present invention throughthe luer ports and analyzed for content of different cells. 70% of thePBMC of the original blood product were recovered from the PBMC layer.This cell suspension only contained 1.1% of the original red bloodcells. The present invention thus can be used for automated preparationof peripheral blood mononuclear cells (second ring) or platelet richplasma (inner ring).

Example 16 Cell Culture

The centrifugation chamber of the present invention can be used forculturing of cells, similarly to cell culture flasks or bags. 3.2E5/mlof the human cell line K562 have been applied to a centrifugationchamber in a volume of 30 ml RPMI1640 cell culture medium supplementedwith 10% fetal calf serum. The chamber was placed in a CO₂ incubator at5% CO₂. Aliquots of the content have been removed from the chamber forcell counting and viability assessment after 24, 48 and 70 hours. Seededcells expanded to 4.1E5/ml, 6.4E5/ml and 9.2E5/ml viable cells at 80%,95% and 95% viability.

Example 17 Method for the Separation of CD133+Cells Using a System ofthe Present Invention

The whole separation procedure of CD133+ cells comprises several stepswith following tasks:

1. Priming of the tubing set with buffer2. Transfer of the bone marrow sample to centrifugation chamber3. Preparation of the bone marrow sample in the centrifugation chamber.4. Magnetic separation of CD133+ cells5. Final volume reduction

In the following, several steps will be described in detail.

1. Priming of the Tubing Set with Buffer

In a first step the tubing set was primed with PEB (phosphate bufferedsaline supplemented with 2 mmol/l EDTA; 0.5% HSA).

2. Transfer of the Bone Marrow Sample to Centrifugation Chamber

After priming of the tubing set the bone marrow sample was transferredfrom the sample bag 312 to the centrifugation chamber 332.

3. Preparation of the Bone Marrow Sample in the Centrifugation Chamber.

The sample preparation in the centrifugation chamber includes severalsteps where supernatant is removed from the chamber. During supernatantremoval only cell free supernatant or supernatant with platelets in caseof the platelet wash should be transferred. It is the aim to avoid whiteblood cells (WBCs) and therefore CD133+ cells to be removed along withthe supernatant. The sample preparation includes following steps:

-   -   generation of plasma    -   reduction of platelets    -   incubation with CD133 Microbeads (Miltenyi Biotec, Germany)    -   removal of unbound CD133 Microbeads

Generation of Plasma:

The plasma generated at the beginning of the sample preparation processserves the following two functions:

-   1. to act as a blocking reagent that blocks the Fc receptors of    monocytes and hinders the Fc part of the CD133 antibodies bound to    the microbeads to attach to monocytes. This would lead to a reduced    purity of the product of the immunomagnetic separation process by    monocytes, and-   2. to act as a supplement for the buffer in which the final cell    product is suspended. This provides a more physiological environment    for the cell product than the used buffer alone. For the generation    and collection of plasma, the sample is centrifuged until the    majority of cellular components of the material as erythrocytes    (RBCs), white blood cells (WBCs) and platelets are pelletized. The    cell free supernatant is the transferred to a container (303) that    acts as a reservoir

For plasma generation, the cell material was centrifuged with 2000 rpmin the centrifugation chamber until all cellular components of thematerial as WBCs, RBCs and platelets are pelletized. The generation ofplasma requires a centrifugation that leads to a supernatant with as lowcell concentrations as possible and therefore a nearly completesedimentation of all cellular blood components. The sedimentation timedepends on the sample volume that is determined automatically during thesample transfer into the centrifugation chamber. The sedimentation timefor different sample volumes is shown in table 2:

TABLE 2 Sedimentation time for different suspension volumes in theplasma generation process Radius of Maximum air/liquid Volume intervalvolume boundary layer Sedimentation time ≦100 ml 100 ml 5.21 cm  8.01min ≈ 8 min 100 ml < V ≦ 125 ml 125 ml 4.99 cm 11.82 min ≈ 12 min 125 ml< V ≦ 150 ml 150 ml 4.77 cm 15.98 min ≈ 16 min 150 ml < V ≦ 175 ml 175ml 4.53 cm 20.57 min ≈ 21 min 175 ml < V ≦ 200 ml 200 ml 4.28 cm 25.69min ≈ 26 min

After centrifugation at 2000 rpm the rotation speed was gently reducedto 1000 rpm with a deceleration rate of 632 rpm/min. Then plasma isremoved with a pump speed of 6 ml/min until air was pumped out of thechamber. The cell free supernatant is then transferred to a containerthat acts as a reservoir. Later, the plasma can be transferred from thecontainer to the site in the process where it is used flow through afilter with 0.2 μm to remove all residual cells that were not removedduring centrifugation.

Reduction of Platelets:

As the plasma generation removes the complete supernatant in thechamber, a residual volume of 70 ml is in the chamber. 150 ml PEB areadded to dilute the suspension resulting in a total volume of 220 ml.The drum is then accelerated to 2000 rpm and the sedimentation executedfor 100 sec in order to pelletize the RBCs and WBCs before the drum wasdecelerated to 1000 rpm with a deceleration rate of 632 rpm/min. Withinthis sedimentation time, only a small portion of the platelets reach thepellet when the deceleration of the chamber starts. Then the completesupernatant is removed at a speed of 6 ml/min. Analysis of the sampleand the removed supernatant showed an experimental determined plateletremoval of 50.8%. The remaining platelets are removed using a Filter(310).

Incubation with CD133 Microbeads

The CD133 microbeads are provided in a glass vials having a septum. Inorder to assure asep-tic conditions, a filter with 0.2 μm pore size(306) is integrated into the branch of the tubing set connected to thevial. The vial is connected to the tubing set by a vented vial adapter(305). When the reagent is pumped to the chamber, the pressure withinthe part of the tubing set in front of the drum is monitored. When thevial runs empty, air moves to the filter. As the filter is wetted by thereagent, the pores are still filled with liquid when the air moves intothe filter. Due to capillary forces the air cannot pass the membrane ofthe filter. As the pump continues to work, the pressure drop is detectedby pressure sensor (329) and the software stops the pump. In this waycombined with a rinsing step to remove residual reagent, the reagent istransferred automatically to the cell suspension in the chamber.

After transfer of plasma for blocking purposes the volume is adjusted tothe labeling volume of 95 ml and the incubation starts. In theconventional sample preparation process the sample preparation bag hasto be put on an orbital shaker and incubated for 30 mm at roomtemperature. The incubation process with the system of the presentinvention is realized as follows:

-   1. The drum is accelerated to a speed of 300 rpm. This makes the    liquid move to the wall of the chamber.-   2. The centrifugation is carried out for 10 seconds.-   3. After that, the drum is stopped. The liquid moves back and has a    horizontally aligned surface.-   4. The chamber continues to stop for 50 seconds.-   5. Steps 1-4 are repeated until the incubation time of 30 mm is    elapsed.

This process leads to an efficient mixing of the liquid in the chamber.

Removal of Unbound Anti-CD133 Microbeads:

The beads wash is performed in order to remove the unbound reagent afterthe labeling and incubation. In the wash process the following steps arerepeated for three times:

-   -   1. PEB is transferred to the centrifuge to dilute the suspension        resulting in a total volume of 230 ml    -   2. Sedimentation of RBCs and WBCs is carried out at 2000 rpm for        140 seconds    -   3. The speed of the chamber is reduced from 2000 rpm to 1000 rpm    -   4. Supernatant is removed completely at a speed of 20 ml/min

The beads wash process will consist of three washing steps and at least97.2% of the unbound reagent is removed.

4. Magnetic Separation of CD133+Cells

After the beads wash process CD133+ cells are separated using a magneticseparation column (CliniMACS column, Miltenyi Biotec GmbH, Germany).Therefore the cell suspension is transferred on the separation columnwith a loading rate of 5 ml/min. The magnetically labeled cells areretained in the magnetized column and separated from the unlabeled cellsby rinsing of the column with PEB. The retained CD133+ cells are elutedby removing the magnetic field from the column. The CD133+ cells arepumped into a loop of the tubing set and reloaded on the separationcolumn. This process was repeated once again and after the thirdreloading process, the CD133+ cells are finally eluted in 20 ml elutionbuffer (see table 3 and FIG. 26). The elution rate is 600 ml/min.

5. Final Volume Reduction

For cardial stem cell therapy, CD133+ cells should be available in asmall final volume at most of 6 ml. Normally, CD133+ cells are finallyeluted from the CliniMACS column in 20 ml elution buffer. For reducingof the final volume, three methods are possible: The final volume ofisolated or enriched CD133+ cells can be reduced by

-   -   1. elution from the column in small volume,    -   2. filtration after magnetic separation, or    -   3. using the AutoMACS column (Miltenyi Biotec GmbH, Germany).        Elution from the Column in Small Volume:

Instead of elution in 20 ml elution buffer, CD133+ cells can be elutedfrom the CliniMACS column in 6 ml elution buffer. The elution rate is600 ml/min. This method was tested by the enrichment of CD133+ cellsfrom bone marrow. Enriched cells were determined by FACS analyses (seetable 4 and FIG. 27).

Filtration after Magnetic Separation

After elution from the CliniMACS separation column in 20 ml elutionbuffer CD133+ cells can be transferred on a filter (Pall IV-5, 0.2 μm orRoweFil 24, 1.2 μm) at a rate of 4 ml/min. Afterwards the cells areeluted in 2 ml from the filter. This method was tested by the enrichmentof CD133+ cells from bone marrow. Enriched cells were determined by FACSanalyses (see table 5 and FIG. 28).

Final Volume Reduction by Using the AutoMACS Column

After elution from the CliniMACS column CD133+ cells can be loaded on anautoMACS column at a rate of 4 ml/min. Afterwards retained CD133+ cellsare eluted in 4 ml from the autoMACS column. This method was tested byenrichment of CD133+ cells from bone marrow. Enriched dells weredetermined by FACS analyses (see table 6 and FIG. 29).

TABLE 3 Direct elution of the CD133+ cells in 20 ml. Start StartingFinal cell volume frequency Final purity CD133− log number CD133+ [ml]CD133+ CD133+ depletion CD133+ yield* 91 0.39% 82.95% 3.4 2.89E6 49.57%86 0.67% 66.99% 2.8 5.72E6 56.67% 129 0.31%  82.6% 3.4 4.11E6 43.42% 570.76% 59.18% 2.3  5.1E6 72.24% 61 0.14% 74.72% 3.2 4.25E5 84.32% 1140.37% 88.19% 3.4 1.79E6 50.08% 77 0.74%  83.5% 3 2.12E6 58.56%

TABLE 4 Direct elution of the CD133+ cells in 6 ml. Start Starting Finalcell volume frequency Final purity CD133− log number CD133+ [ml] CD133+CD133+ depletion CD133+ yield* 87 0.67% 62.79% 2.7 2.78E6 51.22% 1290.31% 81.12% 3.6 1.25E6  33.6% 128 0.66% 57.25% 2.6 2.31E6 26.86%

TABLE 5 Concentration of the CD133 + cells by filtration after magneticseparation. Start volume/ final Starting Final Final cell volumefrequency purity CD133− log number CD133+ [ml] CD133+ CD133+ depletionCD133+ yield* 91/3.79 0.39% 66.08% 1.2 6.27E5 25.3% 86/3.67 0.67%  60.1%1.5 1.59E6 31.6% 129/3.66  0.31% 76.04% 1.2 8.18E5 26.6%

TABLE 6 Concentration of the CD133 + cells by using the AutoMACS column.Start volume/ Starting Final CD133− Final cell final frequency puritylog number CD133+ volume [ml] CD133+ CD133+ depletion CD133+ yield*57/2.78 0.76%  71.3% 0.9 2.35E6 39.9% 61/5.68 0.14% 82.84% 0.4 1.73E543.1% 114/5.64  0.37% 93.57% 0.7 8.51E5 28.6% (*calculated in relationto CD133+ cells recovered in all bags)

1. An apparatus configured for fractionating a blood or bone marrowsample, the apparatus comprising: a) a first connecting memberconfigured to receive the sample and deliver the sample to a rotatablecontainer, b) said rotatable container, wherein the rotatable containercontains at least one processing chamber and is configured to rotateabout an axis and thereby to apply centrifugal force to a blood or bonemarrow sample in the chamber so as to separate different cell types fromthe sample according to a first cell characteristic; c) a secondconnecting member configured to receive a selected subset of cells thathas been separated from the sample by the rotatable container and todeliver the subset to a cell separation column in a column holder; d)said column holder, which is configured to reversibly secure a cellseparation column to the apparatus such that cells can be delivered tothe cell separation column by the second connecting member; and e) athird connecting member configured to recover cells that have beenselected by a cell separation column in the holder and to deliver theselected cells to a product collection container; wherein the apparatusis constructed so that a sample can be processed from the firstconnecting member through the rotatable container and through a cellseparation column in the holder and a product thereof can be deliveredinto the product collection container while maintaining sterilitythroughout all the aforelisted components.
 2. The apparatus of claim 1,wherein the apparatus contains a second holder that is configured toreversibly secure the rotatable container to the apparatus such that thecontainer is operatively connected to a centrifuge drive, but isremovable for sterilization or disposal.
 3. The apparatus of claim 1,wherein the rotatable container comprises a detection window that ispositioned substantially perpendicular to the axis of rotation such thata detector can assess what is contained in the chamber radially throughand along the detection window.
 4. The apparatus of claim 3, wherein aprism or a mirror is positioned radially along the detection window. 5.The apparatus of claim 1, wherein the first connecting member isconnected to the processing chamber of the rotatable container by way ofa central line that passes through rotational seals and along the axis.6. The apparatus of claim 1, wherein the first and the second connectingmembers are connected to the processing chamber of the rotatablecontainer by way of an inner line and an outer line that both passthrough rotational seals and along the axis.
 7. The apparatus of claim3, further comprising a detector that is positioned so as to receivelight that has been provided by a light source and has passed through aportion of the sample along a radial cross-section of the rotatablecontainer.
 8. The apparatus of claim 7, configured so that uponprocessing of a blood sample in the rotatable container, the detectorcan detect an outer ring of erythrocytes and granulocytes, a ring ofperipheral blood mononuclear cells, and an inner ring of platelets. 9.The apparatus of claim 1, whereby the rotatable container comprises aninlet and an outlet port configured for exchange of air or gas insidethe chamber so as to support culturing of cells in the chamber understerile conditions without stopping the centrifuge.
 10. The apparatus ofclaim 1, wherein the rotatable container comprises a plurality ofsurfaces perpendicular to the rotational axis upon which cells may becultured.
 11. The apparatus of claim 1, wherein the first characteristicis cell density.
 12. The apparatus of claim 11, wherein the firstcharacteristic is affinity for a surface, and the rotatable container isconstructed such that such that cells adhering to the surfaces may beseparated while the chamber is rotating from cells with lesser affinityfor the surfaces.
 13. The apparatus of claim 1, comprising fluidcircuitry configured to distribute a plurality of different cellfractions from the rotatable container and/or the cell separation columneach to a different cell collection container.
 14. The apparatus ofclaim 1, comprising fluid circuitry configured to pass cells multipletimes over a sample separation column in the holder.
 15. A systemcomprising the apparatus of claim 1 with a cell separation columnsecured by and connected in the column holder, wherein the cellseparation column is configured to separate hematopoietic cells from therotatable container according to a characteristic of a subset of cellsin a sample, or a label that is attached selectively to a subset ofcells in the sample.
 16. The system of claim 15, wherein the cellseparation column is a magnetic separation column configured so thatcells labeled with a magnetic label will be retained by the column, andcells that are not so labeled will pass through the column.
 17. Thesystem of claim 5, wherein the cell separation column is configured witha ferromagnetic matrix that can be rendered magnetic to retain cellslabeled with a magnetic particle, and then can be rendered non-magneticto release cells that have been retained.
 18. A method for fractionatinga cell sample using the apparatus of claim 1, comprising: a) securing acell separation column to the holder; b) delivering the sample throughthe first connecting member into the rotatable container, c) causing therotatable container to rotate, thereby applying centrifugal force to thesample so as to separate different cell types from the sample accordingto a first characteristic; d) processing a cell type separated by therotatable container in the cell separation column so as to select cellstherefrom according to a second characteristic; and e) recovering cellsfrom the apparatus selected as having both the first and the secondcharacteristics.
 19. The method of claim 18, wherein the firstcharacteristic is a cell density of a particular hematopoetic cell type.20. The method of claim 18, wherein the second characteristic comprisesthe presence or absence of a cell-surface marker selected from CD34,CD133, CD14, CD304, CD4, and CD8.
 21. An apparatus configured forfractionating a cell sample, the apparatus comprising: a) a firstconnector configured for sterile connection of a sample container; b) asample processing unit configured to reversibly receive a rotatablecontainer, comprising: i) a first holder sized and shaped to reversiblysecure the rotatable container; ii) a drive configured to rotate therotatable container when secured in the first holder, thereby applyingcentrifugal force to a blood or bone marrow sample in the container soas to separate different cell types in the sample according to a firstcell characteristic; iii) a first inlet port configured to deliver afirst cell population into the rotatable container for separation; andiv) a first outlet port configured to receive a selected subset of cellsfrom the first cell population that have been separated by the rotatablecontainer; c) a sample separation unit configured to reversibly receivea cell separation column, the second holder comprising: i) a secondholder sized and shaped to reversibly secure the cell separation column;ii) a second inlet port configured to deliver a second cell populationonto a cell separation column secured in the second holder forseparation; and iii) a second outlet port configured to receive a subsetof cells that have been selected from the second cell population by thecell separation column; d) a second connector configured for sterileconnection to product collection container; wherein the apparatus isconfigured so that when a cell sample is supplied through the firstconnector, the sample can be processed by a rotatable container that issecured in the first holder, then processed by a cell separation columnthat is secured in the second holder, following which a producttherefrom can be delivered though the second connector into a samplecollection container, throughout all of which cells processed by thesystem are kept in a sterile environment.
 22. A system comprising theapparatus of claim 21 with a rotatable container secured by andconnected to the first holder and a cell separation column secured byand connected to the second holder, wherein the cell separation columnis configured to separate hematopoietic cells received from therotatable container into one or more subsets according to acharacteristic of cells in the sample, or a label that is attachedselectively to a subset of cells in the sample.
 23. The system of claim1, wherein the rotatable container comprises an axis of rotation, andfirst inlet port and the first outlet port are connected to a processingchamber in the rotatable container by way of an inner line and an outerline that both pass through rotational seals and along the axis ofrotation.
 24. A method for fractionating a blood or bone marrow sampleusing the apparatus of claim 23, comprising: a) securing a rotatablecontainer to the first holder b) securing a cell separation column tothe second holder; c) delivering the sample to the sample preparationunit, d) causing the rotatable container to rotate, thereby applyingcentrifugal force to the sample so as to separate different cell typesfrom the sample according to a first characteristic; then e) deliveringa cell type separated in step d) to the sample processing unit; f)processing the cells delivered in step e) in the cell separation columnso as to select a subset of cells therefrom according to a secondcharacteristic; and g) recovering cells from the apparatus selected ashaving both the first and the second characteristics.
 25. The method ofclaim 24, wherein the second characteristic is presence of a detectablelabel.